Coronavirus vaccine

ABSTRACT

The present invention is directed to a nucleic acid suitable for use in treatment or prophylaxis of an infection with a coronavirus, preferably with a Coronavirus SARS-CoV-2, or a disorder related to such an infection, preferably COVID-19. The present invention is also directed to compositions, polypeptides, and vaccines. The compositions and vaccines preferably comprise at least one of said nucleic acid sequences, preferably nucleic acid sequences in association a lipid nanoparticle (LNP). The invention is also directed to first and second medical uses of the nucleic acid, the composition, the polypeptide, the combination, the vaccine, and the kit, and to methods of treating or preventing a coronavirus infection, preferably a Coronavirus infection.

The present application is a continuation of U.S. application Ser. No.17/276,788, filed Mar. 16, 2021, which is a national phase applicationunder 35 U.S.C. § 371 of International Application No.PCT/EP2021/052455, filed Feb. 3, 2021, which claims priority to U.S.Provisional Application No. 63/129,395, filed Dec. 22, 2020, U.S.Provisional Application No. 63/119,390, filed Nov. 30, 2020, U.S.Provisional Application No. 63/113,159, filed Nov. 12, 2020, U.S.Provisional Application No. 63/112,106, filed Nov. 10, 2020,International Application No. PCT/EP2020/080713, filed Nov. 2, 2020,International Application No. PCT/EP2020/079973, filed Oct. 23, 2020,International Application No. PCT/EP2020/079831, filed Oct. 22, 2020,International Application No. PCT/EP2020/065091, filed May 29, 2020,International Application No. PCT/EP2020/059687, filed Apr. 3, 2020, andInternational Application No. PCT/EP2020/052775, filed Feb. 4, 2020, theentire contents of each of which are hereby incorporated by reference.

The sequence listing that is contained in the file named“CRVCP0311USC3_ST25.txt”, which is 166,970,048 byes (as measured inMicrosoft Windows®) and was created on Dec. 9, 2021, is filedconcurrently herewith on compact discs by Priority Express Mail and isincorporated by reference herein.

INTRODUCTION

The present invention is inter alia directed to a nucleic acid suitablefor use in treatment or prophylaxis of an infection with a coronavirus,preferably with a coronavirus SARS-CoV-2, or a disorder related to suchan infection, preferably COVID-19. The present invention is alsodirected to compositions, polypeptides, and vaccines. The compositionsand vaccines preferably comprise at least one of said nucleic acidsequences, preferably nucleic acid sequences in association with apolymeric carrier, a polycationic protein or peptide, or a lipidnanoparticle (LNP). The invention is also directed to first and secondmedical uses of the nucleic acid, the composition, the polypeptide, thevaccine, and the kit, and to methods of treating or preventing acoronavirus infection, preferably a SARS-CoV-2 infection.

Coronaviruses are enveloped, positive single stranded RNA viruses of theCoronaviridae family.

Their representatives cause very various diseases in differentvertebrates such as mammals, birds and fish.

Coronaviruses are genetically highly variable, and individual virusspecies can also infect several host species by overcoming the speciesbarrier. Such transfers have resulted in infections in humans with theSARS-associated coronavirus (SARS-CoV) and with the Middle Eastrespiratory syndrome coronavirus (MERS-CoV). The coronavirus epidemicthat started in the Chinese city of Wuhan at the turn of 2019/2020 isattributed to a previously unknown coronavirus, which was given thepreliminary names nCoV-2019 or Wuhan Human Coronavirus (WHCV); later thevirus was given the official name SARS-CoV-2.

Typical symptoms of a SARS-CoV-2 caused virus infection, also referredto as COVID-19 disease (Coronavirus disease 2019), include fever, cough,shortness of breath, pneumonia and gastrointestinal symptoms (e.g.diarrhoea). Severe illness can lead to respiratory failure that requiresmechanical ventilation and support in an intensive care unit. On 30th ofJan. 2020, the world health organization (WHO) declared a global healthemergency over that novel coronavirus outbreak. On March 11, the WHOdeclared COVID-19 a pandemic, pointing to the over 118,000 cases of thecoronavirus illness in over 110 countries and territories around theworld and the sustained risk of further global spread. By end of March2020, there were more than 800,000 confirmed cases of a SARS-CoV-2infection, spreading across almost every country in the world, with morethan 40,000 COVID-19 associated deaths.

At present, no vaccine or specific treatment is available for aSARS-CoV-2 infection and/or COVID-19 disease.

Patients diagnosed with a SARS-CoV-2 infection merely receive supportivetreatment based on the individual's symptoms and clinical condition. Dueto the substantial risk of a severe global pandemic, there is an urgentneed for a safe and effective treatment or prophylaxis of SARS-CoV-2infections. In particular, a vaccine is needed to protect the elderlypopulation where high mortality rates have been observed.

Nucleic acid based vaccination, including DNA or RNA, represents apromising technique for novel vaccines against emerging viruses. Nucleicacids can be genetically engineered and administered to a human subject.Transfected cells directly produce the encoded antigen (e.g. provided bya DNA or an RNA, in particular an mRNA), which results in protectiveimmunological responses.

A pivotal role for virus-specific memory T-cells in broad and long-termprotection against SARS-CoV infection has been elucidated (see e.g.Channappanavar, Rudragouda, et al. “Virus-specific memory CD8 T cellsprovide substantial protection from lethal severe acute respiratorysyndrome coronavirus infection.” Journal of virology 88.19 (2014):11034-11044). Virus-specific CD8 T cells are e.g. required for pathogenclearance and for mediating protection after viral challenge. Aneffective SARS-CoV-2 vaccine should therefore not only induce strongfunctional humoral immune responses, but also induce SARS-CoV-2 specificCD8+ T-cell and CD4+ T-cell responses.

Therefore, it is the object of the underlying invention to provide anucleic acid based vaccine for coronavirus infections, in particular forSARS-CoV-2 infections. It is a further object of the present inventionto provide an effective coronavirus vaccine, which can be stored andtransported without cold chain and which enables rapid and scalablecoronavirus vaccine production.

As further defined in the claims and the underlying description, theseobjects are inter alia solved by providing a nucleic acid, e.g. an RNAor a DNA, comprising at least one coding sequence encoding at least oneantigenic peptide or protein derived from a coronavirus SARS-CoV-2.

Further, it would be desirable that such a nucleic acid, or e.g. acomposition/vaccine comprising said nucleic acid has at least some ofthe following advantageous features:

-   -   Translation of the nucleic acid at the site of        injection/vaccination (e.g. muscle);    -   Very efficient induction of antigen-specific immune responses        against the encoded SARS-CoV-2 protein at a very low dosage and        dosing regimen;    -   Suitability for vaccination of infants and/or newborns or the        elderly, in particular the elderly;    -   Suitability of the composition/vaccine for intramuscular        administration;    -   Induction of specific and functional humoral immune response        against coronavirus, in e.g. SARS-CoV-2;    -   Induction of broad, functional cellular T-cell responses against        coronavirus, in e.g. SARS-CoV-2;    -   Induction of specific B-cell memory against coronavirus, in e.g.        SARS-CoV-2;    -   Induction of functional antibodies that can effectively        neutralize the virus, e.g. SARS-CoV-2;    -   Induction of functional antibodies that can effectively        neutralize emerging variants of SARS-CoV-2;    -   Eliciting of mucosal IgA immunity by inducing of mucosal IgA        antibodies,    -   Induction of a well-balanced B cell and T cell responses;    -   Induction of protective immunity against coronavirus infection,        e.g. against SARS-CoV-2 or emerging variants thereof;    -   Fast onset of immune protection against coronavirus, in e.g.        SARS-CoV-2;    -   Longevity of the induced immune responses against coronavirus,        in e.g. SARS-CoV-2;    -   No enhancement of a SARS-CoV-2 infection due to vaccination or        immunopathological effects;    -   No antibody dependent enhancement (ADE) caused by the nucleic        acid based SARS-CoV-2 vaccine;    -   No excessive induction of systemic cytokine or chemokine        response after application of the vaccine, which could lead to        an undesired high reactogenicity upon vaccination;    -   Well tolerability, no side-effects, non-toxicity of the vaccine;    -   Advantageous stability characteristics of the nucleic acid-based        vaccine;    -   Speed, adaptability, simplicity and scalability of coronavirus        vaccine production;    -   Advantageous vaccination regimen that only requires one or two        vaccination for sufficient protection.    -   Advantageous vaccination regimen that only requires a low dose        of the composition/vaccine for sufficient protection.

Definitions

For the sake of clarity and readability, the following definitions areprovided. Any technical feature mentioned for these definitions may beread on each and every embodiment of the invention. Additionaldefinitions and explanations may be specifically provided in the contextof these embodiments.

Percentages in the context of numbers should be understood as relativeto the total number of the respective items. In other cases, and unlessthe context dictates otherwise, percentages should be understood aspercentages by weight (wt.-%).

About: The term “about” is used when determinants or values do not needto be identical, i.e. 100% the same. Accordingly, “about” means, that adeterminant or values may diverge by 0.1% to 20%, preferably by 0.1% to10%; in particular, by 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%. The skilled personwill know that e.g. certain parameters or determinants may slightly varybased on the method how the parameter was determined. For example, if acertain determinants or value is defined herein to have e.g. a length of“about 1000 nucleotides”, the length may diverge by 0.1% to 20%,preferably by 0.1% to 10%; in particular, by 0.5%, 1%, 2%, 3%, 4%, 5%,6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%.Accordingly, the skilled person will know that in that specific example,the length may diverge by 1 to 200 nucleotides, preferably by 1 to 200nucleotides; in particular, by 5, 10, 20, 30, 40, 50, 60, 70, 80, 90,100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 nucleotides.

Adaptive immune response: The term “adaptive immune response” as usedherein will be recognized and understood by the person of ordinary skillin the art, and is e.g. intended to refer to an antigen-specificresponse of the immune system (the adaptive immune system). Antigenspecificity allows for the generation of responses that are tailored tospecific pathogens or pathogen-infected cells. The ability to mountthese tailored responses is usually maintained in the body by “memorycells” (B-cells). In the context of the invention, the antigen isprovided by the nucleic acid (e.g. an RNA or a DNA) encoding at leastone antigenic peptide or protein derived from coronavirus, preferablyfrom SARS-CoV-2 (nCoV-2019).

Antigen: The term “antigen” as used herein will be recognized andunderstood by the person of ordinary skill in the art, and is e.g.intended to refer to a substance which may be recognized by the immunesystem, preferably by the adaptive immune system, and is capable oftriggering an antigen-specific immune response, e.g. by formation ofantibodies and/or antigen-specific T cells as part of an adaptive immuneresponse. Typically, an antigen may be or may comprise a peptide orprotein, which may be presented by the MHC to T-cells. Also fragments,variants and derivatives of peptides or proteins derived from e.g. spikeprotein (S) of coronavirus, preferably from SARS-CoV-2 (nCoV-2019)comprising at least one epitope are understood as antigens in thecontext of the invention. In the context of the present invention, anantigen may be the product of translation of a provided nucleic acid asspecified herein.

Antigenic peptide or protein: The term “antigenic peptide or protein” or“immunogenic peptide or protein” will be recognized and understood bythe person of ordinary skill in the art, and is e.g. intended to referto a peptide, protein derived from a (antigenic or immunogenic) proteinwhich stimulates the body's adaptive immune system to provide anadaptive immune response. Therefore an antigenic/immunogenic peptide orprotein comprises at least one epitope (as defined herein) or antigen(as defined herein) of the protein it is derived from (e.g., spikeprotein (S) of coronavirus, preferably from SARS-CoV-2 (nCoV-2019)).

Cationic: Unless a different meaning is clear from the specific context,the term “cationic” means that the respective structure bears a positivecharge, either permanently or not permanently, but in response tocertain conditions such as pH. Thus, the term “cationic” covers both“permanently cationic” and “cationisable”.

Cationisable: The term “cationisable” as used herein means that acompound, or group or atom, is positively charged at a lower pH anduncharged at a higher pH of its environment. Also in non-aqueousenvironments where no pH value can be determined, a cationisablecompound, group or atom is positively charged at a high hydrogen ionconcentration and uncharged at a low concentration or activity ofhydrogen ions. It depends on the individual properties of thecationisable or polycationisable compound, in particular the pKa of therespective cationisable group or atom, at which pH or hydrogen ionconcentration it is charged or uncharged. In diluted aqueousenvironments, the fraction of cationisable compounds, groups or atomsbearing a positive charge may be estimated using the so-calledHenderson-Hasselbalch equation, which is well-known to a person skilledin the art. E.g., in some embodiments, if a compound or moiety iscationisable, it is preferred that it is positively charged at a pHvalue of about 1 to 9, preferably 4 to 9, 5 to 8 or even 6 to 8, morepreferably of a pH value of or below 9, of or below 8, of or below 7,most preferably at physiological pH values, e.g. about 7.3 to 7.4, i.e.under physiological conditions, particularly under physiological saltconditions of the cell in vivo. In other embodiments, it is preferredthat the cationisable compound or moiety is predominantly neutral atphysiological pH values, e.g. about 7.0-7.4, but becomes positivelycharged at lower pH values. In some embodiments, the preferred range ofpKa for the cationisable compound or moiety is about 5 to about 7.

Coding sequence/coding region: The terms “coding sequence” or “codingregion” and the corresponding abbreviation “cds” as used herein will berecognized and understood by the person of ordinary skill in the art,and are e.g. intended to refer to a sequence of several nucleotidetriplets, which may be translated into a peptide or protein. A codingsequence in the context of the present invention may be a DNA sequence,preferably an RNA sequence, consisting of a number of nucleotides thatmay be divided by three, which starts with a start codon and whichpreferably terminates with a stop codon.

Derived from: The term “derived from” as used throughout the presentspecification in the context of a nucleic acid, i.e. for a nucleic acid“derived from” (another) nucleic acid, means that the nucleic acid,which is derived from (another) nucleic acid, shares e.g. at least 60%,70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the nucleicacid from which it is derived. The skilled person is aware that sequenceidentity is typically calculated for the same types of nucleic acids,i.e. for DNA sequences or for RNA sequences. Thus, it is understood, ifa DNA is “derived from” an RNA or if an RNA is “derived from” a DNA, ina first step the RNA sequence is converted into the corresponding DNAsequence (in particular by replacing the uracils (U) by thymidines (T)throughout the sequence) or, vice versa, the DNA sequence is convertedinto the corresponding RNA sequence (in particular by replacing the T byU throughout the sequence). Thereafter, the sequence identity of the DNAsequences or the sequence identity of the RNA sequences is determined.Preferably, a nucleic acid “derived from” a nucleic acid also refers tonucleic acid, which is modified in comparison to the nucleic acid fromwhich it is derived, e.g. in order to increase RNA stability evenfurther and/or to prolong and/or increase protein production. In thecontext of amino acid sequences (e.g. antigenic peptides or proteins)the term “derived from” means that the amino acid sequence, which isderived from (another) amino acid sequence, shares e.g. at least 60%,70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with theamino acid sequence from which it is derived.

Epitope: The term “epitope” (also called “antigen determinant” in theart) as used herein will be recognized and understood by the person ofordinary skill in the art, and is e.g. intended to refer to T cellepitopes and B cell epitopes. T cell epitopes or parts of the antigenicpeptides or proteins and may comprise fragments preferably having alength of about 6 to about 20 or even more amino acids, e.g. fragmentsas processed and presented by MHC class I molecules, preferably having alength of about 8 to about 10 amino acids, e.g. 8, 9, or 10, (or even11, or 12 amino acids), or fragments as processed and presented by MHCclass II molecules, preferably having a length of about 13 to about 20or even more amino acids. These fragments are typically recognized by Tcells in form of a complex consisting of the peptide fragment and an MHCmolecule, i.e. the fragments are typically not recognized in theirnative form. B cell epitopes are typically fragments located on theouter surface of (native) protein or peptide antigens, preferably having5 to 15 amino acids, more preferably having 5 to 12 amino acids, evenmore preferably having 6 to 9 amino acids, which may be recognized byantibodies, i.e. in their native form. Such epitopes of proteins orpeptides may furthermore be selected from any of the herein mentionedvariants of such proteins or peptides. In this context epitopes can beconformational or discontinuous epitopes which are composed of segmentsof the proteins or peptides as defined herein that are discontinuous inthe amino acid sequence of the proteins or peptides as defined hereinbut are brought together in the three-dimensional structure orcontinuous or linear epitopes which are composed of a single polypeptidechain.

Fragment: The term “fragment” as used throughout the presentspecification in the context of a nucleic acid sequence (e.g. RNA or aDNA) or an amino acid sequence may typically be a shorter portion of afull-length sequence of e.g. a nucleic acid sequence or an amino acidsequence. Accordingly, a fragment, typically, consists of a sequencethat is identical to the corresponding stretch within the full-lengthsequence. A preferred fragment of a sequence in the context of thepresent invention, consists of a continuous stretch of entities, such asnucleotides or amino acids corresponding to a continuous stretch ofentities in the molecule the fragment is derived from, which representsat least 40%, 50%, 60%, 70%, 80%, 90%, 95% of the total (i.e.full-length) molecule from which the fragment is derived (e.g. spikeprotein (S) of coronavirus, preferably from SARS-CoV-2 (nCoV-2019)). Theterm “fragment” as used throughout the present specification in thecontext of proteins or peptides may, typically, comprise a sequence of aprotein or peptide as defined herein, which is, with regard to its aminoacid sequence, N-terminally and/or C-terminally truncated compared tothe amino acid sequence of the original protein. Such truncation maythus occur either on the amino acid level or correspondingly on thenucleic acid level. A sequence identity with respect to such a fragmentas defined herein may therefore preferably refer to the entire proteinor peptide as defined herein or to the entire (coding) nucleic acidmolecule of such a protein or peptide. Fragments of proteins or peptidesmay comprise at least one epitope of those proteins or peptides.

Heterologous: The terms “heterologous” or “heterologous sequence” asused throughout the present specification in the context of a nucleicacid sequence or an amino acid sequence refers to a sequence (e.g. RNA,DNA, amino acid) has to be understood as a sequence that is derived fromanother gene, another allele, or e.g. another species or virus. Twosequences are typically understood to be “heterologous” if they are notderivable from the same gene or from the same allele. I.e., althoughheterologous sequences may be derivable from the same organism or virus,in nature, they do not occur in the same nucleic acid or protein.

Humoral immune response: The terms “humoral immunity” or “humoral immuneresponse” will be recognized and understood by the person of ordinaryskill in the art, and are e.g. intended to refer to B-cell mediatedantibody production and optionally to accessory processes accompanyingantibody production. A humoral immune response may be typicallycharacterized, e.g. by Th2 activation and cytokine production, germinalcenter formation and isotype switching, affinity maturation and memorycell generation. Humoral immunity may also refer to the effectorfunctions of antibodies, which include pathogen and toxinneutralization, classical complement activation, and opsonin promotionof phagocytosis and pathogen elimination.

Identity (of a sequence): The term “identity” as used throughout thepresent specification in the context of a nucleic acid sequence or anamino acid sequence will be recognized and understood by the person ofordinary skill in the art, and is e.g. intended to refer to thepercentage to which two sequences are identical. To determine thepercentage to which two sequences are identical, e.g. nucleic acidsequences or amino acid (aa) sequences as defined herein, preferably theaa sequences encoded by the nucleic acid sequence as defined herein orthe aa sequences themselves, the sequences can be aligned in order to besubsequently compared to one another. Therefore, e.g. a position of afirst sequence may be compared with the corresponding position of thesecond sequence. If a position in the first sequence is occupied by thesame residue as is the case at a position in the second sequence, thetwo sequences are identical at this position. If this is not the case,the sequences differ at this position. If insertions occur in the secondsequence in comparison to the first sequence, gaps can be inserted intothe first sequence to allow a further alignment. If deletions occur inthe second sequence in comparison to the first sequence, gaps can beinserted into the second sequence to allow a further alignment. Thepercentage to which two sequences are identical is then a function ofthe number of identical positions divided by the total number ofpositions including those positions which are only occupied in onesequence. The percentage to which two sequences are identical can bedetermined using an algorithm, e.g. an algorithm integrated in the BLASTprogram.

Immunogen, immunogenic: The terms “immunogen” or “immunogenic” will berecognized and understood by the person of ordinary skill in the art,and are e.g. intended to refer to a compound that is able tostimulate/induce an immune response. Preferably, an immunogen is apeptide, polypeptide, or protein. An immunogen in the sense of thepresent invention is the product of translation of a provided nucleicacid, comprising at least one coding sequence encoding at least oneantigenic peptide, protein derived from spike protein (S) of SARS-CoV-2(nCoV-2019) as defined herein. Typically, an immunogen elicits anadaptive immune response.

Immune response: The term “immune response” will be recognized andunderstood by the person of ordinary skill in the art, and is e.g.intended to refer to a specific reaction of the adaptive immune systemto a particular antigen (so called specific or adaptive immune response)or an unspecific reaction of the innate immune system (so calledunspecific or innate immune response), or a combination thereof.

Immune system: The term “immune system” will be recognized andunderstood by the person of ordinary skill in the art, and is e.g.intended to refer to a system of the organism that may protect theorganisms from infection. If a pathogen succeeds in passing a physicalbarrier of an organism and enters this organism, the innate immunesystem provides an immediate, but non-specific response. If pathogensevade this innate response, vertebrates possess a second layer ofprotection, the adaptive immune system. Here, the immune system adaptsits response during an infection to improve its recognition of thepathogen. This improved response is then retained after the pathogen hasbeen eliminated, in the form of an immunological memory, and allows theadaptive immune system to mount faster and stronger attacks each timethis pathogen is encountered. According to this, the immune systemcomprises the innate and the adaptive immune system. Each of these twoparts typically contains so called humoral and cellular components.

Innate immune system: The term “innate immune system” (also known asnon-specific or unspecific immune system) will be recognized andunderstood by the person of ordinary skill in the art, and is e.g.intended to refer to a system typically comprising the cells andmechanisms that defend the host from infection by other organisms in anon-specific manner. This means that the cells of the innate system mayrecognize and respond to pathogens in a generic way, but unlike theadaptive immune system, it does not confer long-lasting or protectiveimmunity to the host. The innate immune system may be activated byligands of pattern recognition receptor e.g. Toll-like receptors,NOD-like receptors, or RIG-I like receptors etc.

Lipidoid compound: A lipidoid compound, also simply referred to aslipidoid, is a lipid-like compound, i.e. an amphiphilic compound withlipid-like physical properties. In the context of the present invention,the term lipid is considered to encompass lipidoid compounds.

Permanently cationic: The term “permanently cationic” as used hereinwill be recognized and understood by the person of ordinary skill in theart, and means, e.g., that the respective compound, or group, or atom,is positively charged at any pH value or hydrogen ion activity of itsenvironment. Typically, the positive charge results from the presence ofa quaternary nitrogen atom. Where a compound carries a plurality of suchpositive charges, it may be referred to as permanently polycationic.

Stabilized RNA: The term “stabilized RNA” refer to an RNA that ismodified such, that it is more stable to disintegration or degradation,e.g., by environmental factors or enzymatic digest, such as by exo- orendonuclease degradation, compared to an RNA without such modification.Preferably, a stabilized RNA in the context of the present invention isstabilized in a cell, such as a prokaryotic or eukaryotic cell,preferably in a mammalian cell, such as a human cell. The stabilizationeffect may also be exerted outside of cells, e.g. in a buffer solutionetc., e.g., for storage of a composition comprising the stabilized RNA.

T-cell responses: The terms “cellular immunity” or “cellular immuneresponse” or “cellular T-cell responses” as used herein will berecognized and understood by the person of ordinary skill in the art,and are for example intended to refer to the activation of macrophages,natural killer cells (NK), antigen-specific cytotoxic T-lymphocytes, andthe release of various cytokines in response to an antigen. In moregeneral terms, cellular immunity is not based on antibodies, but on theactivation of cells of the immune system. Typically, a cellular immuneresponse may be characterized e.g. by activating antigen-specificcytotoxic T-lymphocytes that are able to induce apoptosis in cells, e.g.specific immune cells like dendritic cells or other cells, displayingepitopes of foreign antigens on their surface.

Variant (of a sequence): The term “variant” as used throughout thepresent specification in the context of a nucleic acid sequence will berecognized and understood by the person of ordinary skill in the art,and is e.g. intended to refer to a variant of a nucleic acid sequencederived from another nucleic acid sequence. E.g., a variant of a nucleicacid sequence may exhibit one or more nucleotide deletions, insertions,additions and/or substitutions compared to the nucleic acid sequencefrom which the variant is derived. A variant of a nucleic acid sequencemay at least 50%, 60%, 70%, 80%, 90%, or 95% identical to the nucleicacid sequence the variant is derived from. The variant is a functionalvariant in the sense that the variant has retained at least 50%, 60%,70%, 80%, 90%, or 95% or more of the function of the sequence where itis derived from. A “variant” of a nucleic acid sequence may have atleast 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% nucleotide identity overa stretch of at least 10, 20, 30, 50, 75 or 100 nucleotide of suchnucleic acid sequence.

The term “variant” as used throughout the present specification in thecontext of proteins or peptides is e.g. intended to refer to a proteinsor peptide variant having an amino acid sequence which differs from theoriginal sequence in one or more mutation(s)/substitution(s), such asone or more substituted, inserted and/or deleted amino acid(s).Preferably, these fragments and/or variants have the same, or acomparable specific antigenic property (immunogenic variants, antigenicvariants). Insertions and substitutions are possible, in particular, atthose sequence positions which cause no modification to thethree-dimensional structure or do not affect the binding region.Modifications to a three-dimensional structure by insertion(s) ordeletion(s) can easily be determined e.g. using CD spectra (circulardichroism spectra). A “variant” of a protein or peptide may have atleast 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% amino acid identity overa stretch of at least 10, 20, 30, 50, 75 or 100 amino acids of suchprotein or peptide. Preferably, a variant of a protein comprises afunctional variant of the protein, which means, in the context of theinvention, that the variant exerts essentially the same, or at least40%, 50%, 60%, 70%, 80%, 90% of the immunogenicity as the protein it isderived from.

Short Description of the Invention

The present invention is based on the inventor's surprising finding thatat least one peptide or protein derived from coronavirus SARS-CoV-2(formerly called nCoV-2019), provided by the coding sequence of anucleic acid, e.g. an RNA, can efficiently be expressed in human cells(Examples 2a, 2b, and 2c). Even more surprising and unexpected, theadministration of a composition comprising said nucleic acid, e.g. saidRNA, induces antigen-specific immune responses against coronavirus, inparticular against coronavirus SARS-CoV-2 (see Example section).

Even more unexpected, the inventors showed that the coding RNA of theinvention induces high levels of functional antibodies, shown by highvirus neutralizing titers (VNTs) and T-cell responses (vaccines inducedouble positive CD4+ and CD8+ T-cells (see e.g. Example 7 and Example10), and even protects hamsters and NHPs from SARS-CoV-2 challengeinfection (see Example 9 and Example 15), indicating that the coding RNAor the composition/vaccine of the invention is therefore suitable foruse as a vaccine, e.g. as a vaccine in human subjects.

Those findings are the basis for the provision of a nucleic acid basedcoronavirus vaccine.

In a first aspect, the present invention provides a nucleic acid for acoronavirus vaccine, preferably a coronavirus SARS-CoV-2 vaccine,wherein said nucleic acid comprises at least one coding sequenceencoding at least one antigenic peptide or protein of an SARS-CoV-2coronavirus, or an immunogenic fragment or immunogenic variant thereof.

In a second aspect, the present invention provides a composition,preferably an immunogenic composition comprising at least one nucleicacid of the first aspect. Suitably, the composition may comprise atleast one nucleic acid, e.g. at least one coding RNA, complexed with,encapsulated in, or associated with one or more lipids, thereby forminglipid nanoparticles.

In a third aspect, the present invention provides antigenic polypeptidesfor a coronavirus vaccine, preferably for an SARS-CoV-2 composition orvaccine.

In a fourth aspect, the present invention provides a coronavirusvaccine, preferably an SARS-CoV-2 vaccine, wherein the vaccine comprisesat least one nucleic acid of the first aspect, or the composition of thesecond aspect, or at least one polypeptide of the third aspect.

In a fifth aspect, the present invention provides a kit or kit of partscomprising at least one nucleic acid of the first aspect, and/or atleast one composition of the second aspect, and/or at least onepolypeptide of the third aspect, and/or at least one vaccine of theforth aspect.

In a sixth aspect, the present invention provides a combinationcomprising at least two separate components, wherein the at least atleast two separate components are selected from two nucleic acids of thefirst aspect, and/or at least two compositions of the second aspect,and/or at least two polypeptides of the third aspect, and/or at leasttwo vaccine of the forth aspect.

Further aspects of the invention concern a method of treating orpreventing coronavirus infection, preferably an SARS-CoV-2 infection ina subject, and first and second medical uses of nucleic acid,compositions, and vaccines. Also provided are methods of manufacturingthe nucleic acid, the composition, or the vaccine.

DETAILED DESCRIPTION OF THE INVENTION

The present application is filed together with a sequence listing inelectronic format, which is part of the description of the presentapplication (WIPO standard ST.25). The information contained in thesequence listing is incorporated herein by reference in its entirety.Where reference is made herein to a “SEQ ID NO”, the correspondingnucleic acid sequence or amino acid (aa) sequence in the sequencelisting having the respective identifier is referred to. For manysequences, the sequence listing also provides additional detailedinformation, e.g. regarding certain structural features, sequenceoptimizations, GenBank (NCBI) or GISAID (epi) identifiers, or additionaldetailed information regarding its coding capacity. In particular, suchinformation is provided under numeric identifier <223> in the WIPOstandard ST.25 sequence listing. Accordingly, information provided undersaid numeric identifier <223> is explicitly included herein in itsentirety and has to be understood as integral part of the description ofthe underlying invention.

Nucleic Acid for a Coronavirus Vaccine:

In a first aspect, the invention relates to a nucleic acid suitable fora coronavirus vaccine.

It has to be noted that specific features and embodiments that aredescribed in the context of the first aspect of the invention, that isthe nucleic acid of the invention, are likewise applicable to the secondaspect (composition of the invention), the third aspect (polypeptide ofthe invention), the forth aspect (vaccine of the invention), the fifthaspect (kit or kit of parts of the invention), or further aspectsincluding medical uses and method of treatments.

Coronaviruses can be classified into the genus Alphacoronavirus,Betacoronavirus, Deltacoronavirus, Gammacoronavirus, and unclassifiedCoronaviruses. Coronaviruses are genetically highly variable, andindividual virus species can also infect several host species byovercoming the species barrier. Human coronaviruses includeSARS-associated coronavirus (SARS-CoV), Middle East respiratory syndromecoronavirus (MERS-CoV), and coronavirus SARS-CoV-2 (previously named“Wuhan Human coronavirus” or nCoV-2019). Accordingly, the nucleic acidmay be suitable for a vaccine against a coronavirus, preferably againsta coronavirus that is a human pathogen, most preferably against thenovel emerging coronavirus SARS-CoV-2 (nCoV-2019).

The terms “nucleic acid” or “nucleic acid molecule” will be recognizedand understood by the person of ordinary skill in the art. The term“nucleic acid” or “nucleic acid molecule” as used herein preferablyrefers to DNA (molecules) or RNA (molecules). It is preferably usedsynonymously with the term polynucleotide. Preferably, a nucleic acid ora nucleic acid molecule is a polymer comprising or consisting ofnucleotide monomers, which are covalently linked to each other byphosphodiester-bonds of a sugar/phosphate-backbone. The term “nucleicacid molecule” also encompasses modified nucleic acid molecules, such asbase-modified, sugar-modified or backbone-modified DNA or RNA moleculesas defined herein.

The nucleic acid of the first aspect, e.g. the DNA or the RNA, may formthe basis for a nucleic acid based composition or vaccine. Generally,protein-based vaccines, or live attenuated vaccines, are suboptimal foruse in developing countries due to their high production costs. Inaddition, protein-based vaccines, or live attenuated vaccines requirelong development times and are not suitable for rapid responses ofpandemic virus outbreaks such as the Coronavirus SARS-CoV-2 outbreak in2019/2020. In contrast, the nucleic acid-based vaccines according to thepresent invention allow very fast and cost-effective production.Therefore, in comparison with known vaccines, vaccine based on theinventive nucleic acid can be produced significantly cheaper and faster,which is very advantageous particularly for use in developing countries.One further advantage of a vaccine based on the inventive nucleic acidmay be its temperature-stability in comparison to protein orpeptide-based vaccines. However, a vaccine based on a polypeptide isalso in the scope of the underlying invention (see e.g. third aspect).

The terms “nucleic acid sequence”, “DNA sequence”, “RNA sequence” willbe recognized and understood by the person of ordinary skill in the art,and e.g. refer to a particular and individual order of the succession ofits nucleotides.

In a preferred embodiment of the first aspect, the nucleic acidcomprises at least one coding sequence encoding at least one antigenicpeptide or protein from a SARS-CoV-2 (nCoV-2019) coronavirus, or animmunogenic fragment or immunogenic variant thereof.

The term “antigenic peptide or protein from a SARS-CoV-2 coronavirus”has to be understood as (i) an antigen that is from a SARS-CoV-2coronavirus which means that the amino acid sequence of the antigenicpeptide or protein (or a fragment thereof) is identical to a SARS-CoV-2coronavirus protein (or a fragment thereof), or (ii) an antigen that isderived from a SARS-CoV-2 coronavirus which means that the amino acidsequence of the antigenic peptide or protein (or a fragment thereof) isnot identical to a corresponding SARS-CoV-2 coronavirus protein (or afragment thereof).

Accordingly, in a preferred embodiment of the first aspect, the nucleicacid comprises at least one coding sequence encoding at least oneantigenic peptide or protein that is or is derived from an SARS-CoV-2(nCoV-2019) coronavirus, or an immunogenic fragment or immunogenicvariant thereof.

The term “antigenic peptide or protein that is or is derived from aSARS-CoV-2 (nCoV-2019) coronavirus” has to be understood as (i) anantigen that “is from a SARS-CoV-2 coronavirus” which means that theamino acid sequence of the antigenic peptide or protein (or a fragmentthereof) is identical in sequence to a SARS-CoV-2 coronavirus protein(or a fragment thereof), or (ii) an antigen that “is derived from aSARS-CoV-2 coronavirus” which means that the amino acid sequence of theantigenic peptide or protein (or a fragment thereof) is not identical toa sequence of a corresponding SARS-CoV-2 coronavirus protein (or afragment thereof).

In preferred embodiments, the nucleic acid comprises at least one codingsequence encoding at least one antigenic peptide or protein that is oris derived from an SARS-CoV-2 (nCoV-2019) coronavirus, or an immunogenicfragment or immunogenic variant thereof, wherein the nucleic acidcomprises at least one heterologous untranslated region (UTR).

The term “untranslated region” or “UTR” or “UTR element” will berecognized and understood by the person of ordinary skill in the art,and are e.g. intended to refer to a part of a nucleic acid moleculetypically located 5′ or 3′ located of a coding sequence. An UTR is nottranslated into protein. An UTR may be part of a nucleic acid, e.g. aDNA or an RNA. An UTR may comprise elements for controlling geneexpression, also called regulatory elements. Such regulatory elementsmay be, e.g., ribosomal binding sites, miRNA binding sites etc.

As used herein, the terms “Human coronavirus 2019”, “Wuhan Humancoronavirus” (WHCV), “nCoV-2019 coronavirus”, “nCoV-2019”, “Wuhanseafood market pneumonia virus”, “Wuhan coronavirus”, “WHCVcoronavirus”, “HCoV-19”, “SARS2”, “COVID-19 virus”, “hCoV-19”,“SARS-CoV-2”, or “coronavirus SARS-CoV-2” may be used interchangeablethroughout the present invention, relating to a new pandemic coronavirusthat has been emerged in the Chinese city of Wuhan at the turn of2019/2020, causing the disease COVID-19. According to the WHO (February2020), the virus is officially termed “SARS-CoV-2”, and the associateddisease is officially termed “COVID-19”.

The virus SARS-CoV-2 belongs to the Coronaviridae, in particular toOrthocoronaviruses, more specifically to the genus Betacoronavirus.Exemplary SARS-CoV-2 coronaviruses are isolates including but notlimited to those provided in List A and B below.

List A: Exemplary SARS-CoV-2 Coronavirus Isolates (EPI/GISAID):

EPI_ISL_402119, EPI_ISL_402120, EPI_ISL_402121, EPI_ISL_402123,EPI_ISL_402124 (hCoV-19/Wuhan/WIV04/2019), EPI_ISL_402125,EPI_ISL_402127, EPI EPI_ISL_402128 (hCoV-19/Wuhan/WIV05/2019; WIV05;SARS-CoV-2/Wuhan/WIV05/2019_EPI_ISL_402128), EPI_ISL_402129,EPI_ISL_402130, EPI_ISL_402131, EPI_ISL_402132, EPI_ISL_403928,EPI_ISL_403929, EPI_ISL_403930, EPI_ISL_403931, EPI_ISL_403932,EPI_ISL_403933, EPI_ISL_403934, EPI_ISL_403935, EPI_ISL_403936,EPI_ISL_403937, EPI_ISL_403962, EPI_ISL_403963, EPI_ISL_404227,EPI_ISL_404228, EPI_ISL_404253, EPI_ISL_404895, EPI_ISL_405839,EPI_ISL_406030, EPI_ISL_406031, EPI_ISL_406034, EPI_ISL_406036,EPI_ISL_406223, EPI_ISL_406531, EPI_ISL_406533, EPI_ISL_406534,EPI_ISL_406535, EPI_ISL_406536, EPI_ISL_406538, EPI_ISL_406592,EPI_ISL_406593, EPI_ISL_406594, EPI_ISL_406595, EPI_ISL_406596,EPI_ISL_406597, EPI_ISL_406798, EPI_ISL_406800, EPI_ISL_406801,EPI_ISL_406844, EPI_ISL_406862, EPI_ISL_406716, EPI_ISL_406717,EPI_ISL_406970, EPI_ISL_406973, EPI_ISL_407071, EPI_ISL_407073,EPI_ISL_407079, EPI_ISL_407084, EPI_ISL_407193, EPI_ISL_407214,EPI_ISL_407215, EPI_ISL_407313, EPI_ISL_407893, EPI_ISL_407894,EPI_ISL_407896, EPI_ISL_407976, EPI_ISL_407987, EPI_ISL_407988,EPI_ISL_408008, EPI_ISL_408009, EPI_ISL_408010, EPI_ISL_408430,EPI_ISL_408431, EPI_ISL_408478, EPI_ISL_408479, EPI_ISL_408480,EPI_ISL_408481, EPI_ISL_408482, EPI_ISL_408484, EPI_ISL_408486,EPI_ISL_408488, EPI_ISL_408489, EPI_ISL_408514, EPI_ISL_408515,EPI_ISL_408665, EPI_ISL_408666, EPI_ISL_408667, EPI_ISL_408668,EPI_ISL_408669, EPI_ISL_408670, EPI_ISL_408976, EPI_ISL_408977,EPI_ISL_409067, EPI_ISL_410044, EPI_ISL_410045, EPI_ISL_410218,EPI_ISL_410301, EPI_ISL_410486, EPI_ISL_410531, EPI_ISL_410532,EPI_ISL_410535, EPI_ISL_410536, EPI_ISL_410537, EPI_ISL_410538,EPI_ISL_410539, EPI_ISL_410540, EPI_ISL_410541, EPI_ISL_410542,EPI_ISL_410713, EPI_ISL_410714, EPI_ISL_410715, EPI_ISL_410716,EPI_ISL_410717, EPI_ISL_410718, EPI_ISL_410719, EPI_ISL_410720,EPI_ISL_410984, EPI_ISL_411060, EPI_ISL_411066, EPI_ISL_411218,EPI_ISL_411219, EPI_ISL_411220, EPI_ISL_411902, EPI_ISL_411915,EPI_ISL_411926, EPI_ISL_411927, EPI_ISL_411929, EPI_ISL_411950,EPI_ISL_411951, EPI_ISL_411952, EPI_ISL_411953, EPI_ISL_411954,EPI_ISL_411955, EPI_ISL_411956, EPI_ISL_411957, EPI_ISL_412026,EPI_ISL_412028, EPI_ISL_412029, EPI_ISL_412030, EPI_ISL_412459,EPI_ISL_412862, EPI_ISL_412869, EPI_ISL_412870, EPI_ISL_412871,EPI_ISL_412872, EPI_ISL_412873, EPI_ISL_412898, EPI_ISL_412899,EPI_ISL_412912, EPI_ISL_412966, EPI_ISL_412967, EPI_ISL_412968,EPI_ISL_412969, EPI_ISL_412970, EPI_ISL_412972, EPI_ISL_412973,EPI_ISL_412974, EPI_ISL_412975, EPI_ISL_412978, EPI_ISL_412979,EPI_ISL_412980, EPI_ISL_412981, EPI_ISL_412982, EPI_ISL_412983,EPI_ISL_413014, EPI_ISL_413015, EPI_ISL_413016, EPI_ISL_413017,EPI_ISL_413018, EPI_ISL_413021, EPI_ISL_413022, EPI_ISL_413023,EPI_ISL_413024, EPI_ISL_413213, EPI_ISL_413214, EPI_ISL_413455,EPI_ISL_413456, EPI_ISL_413457, EPI_ISL_413458, EPI_ISL_413459,EPI_ISL_413485, EPI_ISL_413486, EPI_ISL_413488, EPI_ISL_413489,EPI_ISL_413490, EPI_ISL_413513, EPI_ISL_413514, EPI_ISL_413515,EPI_ISL_413516, EPI_ISL_413518, EPI_ISL_413519, EPI_ISL_413520,EPI_ISL_413521, EPI_ISL_413522, EPI_ISL_413523, EPI_ISL_413555,EPI_ISL_413557, EPI_ISL_413558, EPI_ISL_413559, EPI_ISL_413560,EPI_ISL_413562, EPI_ISL_413563, EPI_ISL_413566, EPI_ISL_413572,EPI_ISL_413573, EPI_ISL_413577, EPI_ISL_413579, EPI_ISL_413580,EPI_ISL_413581, EPI_ISL_413582, EPI_ISL_413583, EPI_ISL_413584,EPI_ISL_413587, EPI_ISL_413589, EPI_ISL_413590, EPI_ISL_413591,EPI_ISL_413592, EPI_ISL_413593, EPI_ISL_413594, EPI_ISL_413595,EPI_ISL_413596, EPI_ISL_413597, EPI_ISL_413598, EPI_ISL_413599,EPI_ISL_413600, EPI_ISL_413602, EPI_ISL_413603, EPI_ISL_413604,EPI_ISL_413606, EPI_ISL_413607, EPI_ISL_413608, EPI_ISL_413609,EPI_ISL_413610, EPI_ISL_413611, EPI_ISL_413612, EPI_ISL_413613,EPI_ISL_413614, EPI_ISL_413615, EPI_ISL_413616, EPI_ISL_413617,EPI_ISL_413618, EPI_ISL_413619, EPI_ISL_413620, EPI_ISL_413621,EPI_ISL_413622, EPI_ISL_413647, EPI_ISL_413648, EPI_ISL_413691,EPI_ISL_413692, EPI_ISL_413693, EPI_ISL_413694, EPI_ISL_413697,EPI_ISL_413711, EPI_ISL_413729, EPI_ISL_413746, EPI_ISL_413748,EPI_ISL_413749, EPI_ISL_413750, EPI_ISL_413751, EPI_ISL_413761,EPI_ISL_413791, EPI_ISL_413809, EPI_ISL_413852, EPI_ISL_413853,EPI_ISL_413854, EPI_ISL_413856, EPI_ISL_413857, EPI_ISL_413858,EPI_ISL_413860, EPI_ISL_413861, EPI_ISL_413862, EPI_ISL_413863,EPI_ISL_413928, EPI_ISL_413931, EPI_ISL_413996, EPI_ISL_413997,EPI_ISL_413999, EPI_ISL_414005, EPI_ISL_414006, EPI_ISL_414007,EPI_ISL_414008, EPI_ISL_414009, EPI_ISL_414011, EPI_ISL_414012,EPI_ISL_414019, EPI_ISL_414020, EPI_ISL_414021, EPI_ISL_414022,EPI_ISL_414023, EPI_ISL_414027, EPI_ISL_414040, EPI_ISL_414041,EPI_ISL_414042, EPI_ISL_414043, EPI_ISL_414044, EPI_ISL_414045,EPI_ISL_414363, EPI_ISL_414366, EPI_ISL_414367, EPI_ISL_414368,EPI_ISL_414369, EPI_ISL_414414, EPI_ISL_414423, EPI_ISL_414428,EPI_ISL_414429, EPI_ISL_414433, EPI_ISL_414435, EPI_ISL_414439,EPI_ISL_414443, EPI_ISL_414445, EPI_ISL_414446, EPI_ISL_414451,EPI_ISL_414457, EPI_ISL_414468, EPI_ISL_414470, EPI_ISL_414476,EPI_ISL_414477, EPI_ISL_414479, EPI_ISL_414480, EPI_ISL_414481,EPI_ISL_414482, EPI_ISL_414483, EPI_ISL_414484, EPI_ISL_414485,EPI_ISL_414487, EPI_ISL_414500, EPI_ISL_414505, EPI_ISL_414509,EPI_ISL_414510, EPI_ISL_414511, EPI_ISL_414517, EPI_ISL_414519,EPI_ISL_414520, EPI_ISL_414521, EPI_ISL_414522, EPI_ISL_414523,EPI_ISL_414524, EPI_ISL_414525, EPI_ISL_414526, EPI_ISL_414527,EPI_ISL_414528, EPI_ISL_414529, EPI_ISL_414530, EPI_ISL_414531,EPI_ISL_414532, EPI_ISL_414534, EPI_ISL_414535, EPI_ISL_414545,EPI_ISL_414546, EPI_ISL_414547, EPI_ISL_414548, EPI_ISL_414549,EPI_ISL_414552, EPI_ISL_414554, EPI_ISL_414555, EPI_ISL_414556,EPI_ISL_414557, EPI_ISL_414558, EPI_ISL_414559, EPI_ISL_414560,EPI_ISL_414561, EPI_ISL_414562, EPI_ISL_414564, EPI_ISL_414565,EPI_ISL_414566, EPI_ISL_414569, EPI_ISL_414571, EPI_ISL_414574,EPI_ISL_414577, EPI_ISL_414578, EPI_ISL_414579, EPI_ISL_414580,EPI_ISL_414586, EPI_ISL_414587, EPI_ISL_414588, EPI_ISL_414589,EPI_ISL_414590, EPI_ISL_414591, EPI_ISL_414592, EPI_ISL_414593,EPI_ISL_414594, EPI_ISL_414595, EPI_ISL_414596, EPI_ISL_414597,EPI_ISL_414600, EPI_ISL_414601, EPI_ISL_414616, EPI_ISL_414617,EPI_ISL_414618, EPI_ISL_414619, EPI_ISL_414620, EPI_ISL_414621,EPI_ISL_414622, EPI_ISL_414623, EPI_ISL_414624, EPI_ISL_414625,EPI_ISL_414626, EPI_ISL_414627, EPI_ISL_414628, EPI_ISL_414629,EPI_ISL_414630, EPI_ISL_414631, EPI_ISL_414632, EPI_ISL_414633,EPI_ISL_414635, EPI_ISL_414637, EPI_ISL_414638, EPI_ISL_414641,EPI_ISL_414642, EPI_ISL_414643, EPI_ISL_414646, EPI_ISL_414648,EPI_ISL_414663, EPI_ISL_414684, EPI_ISL_414685, EPI_ISL_414686,EPI_ISL_414687, EPI_ISL_414688, EPI_ISL_414689, EPI_ISL_414690,EPI_ISL_414691, EPI_ISL_414692, EPI_ISL_414936, EPI_ISL_414937,EPI_ISL_414938, EPI_ISL_414940, EPI_ISL_414941, EPI_ISL_415105,EPI_ISL_415128, EPI_ISL_415129, EPI_ISL_415136, EPI_ISL_415141,EPI_ISL_415142, EPI_ISL_415147, EPI_ISL_415150, EPI_ISL_415151,EPI_ISL_415152, EPI_ISL_415153, EPI_ISL_415154, EPI_ISL_415155,EPI_ISL_415156, EPI_ISL_415157, EPI_ISL_415158, EPI_ISL_415159,EPI_ISL_415710, EPI_ISL_416426, EPI_ISL_416457, EPI_ISL_416481,EPI_ISL_416489, EPI_ISL_416491, EPI_ISL_416492, EPI_ISL_416514,EPI_ISL_416515, EPI_ISL_416516, EPI_ISL_416517, EPI_ISL_416518,EPI_ISL_416538, EPI_ISL_416539, EPI_ISL_416683, EPI_ISL_416685,EPI_ISL_416704, EPI_ISL_416711, EPI_ISL_416713, EPI_ISL_416715,EPI_ISL_416717, EPI_ISL_416744, EPI_ISL_416830, EPI_ISL_416831,EPI_ISL_416832, EPI_ISL_417020, EPI_ISL_417021, EPI_ISL_417022,EPI_ISL_417023, EPI_ISL_417024, EPI_ISL_417025, EPI_ISL_417026,EPI_ISL_417027, EPI_ISL_417028, EPI_ISL_417034, EPI_ISL_417200,EPI_ISL_417201, EPI_ISL_417202, EPI_ISL_417203, EPI_ISL_417204,EPI_ISL_417374, EPI_ISL_417375, EPI_ISL_417376, EPI_ISL_417377,EPI_ISL_417379, EPI_ISL_417382, EPI_ISL_417408, EPI_ISL_417409,EPI_ISL_417410, EPI_ISL_417411, EPI_ISL_417412, EPI_ISL_417413,EPI_ISL_417420, EPI_ISL_417435, EPI_ISL_417436, EPI_ISL_417437,EPI_ISL_417438, EPI_ISL_417439, EPI_ISL_417440, EPI_ISL_417441,EPI_ISL_417442, EPI_ISL_417467, EPI_ISL_417468, EPI_ISL_417504,EPI_ISL_417505, EPI_ISL_417506, EPI_ISL_417507, EPI_ISL_417508,EPI_ISL_417509, EPI_ISL_417510, EPI_ISL_417512, EPI_ISL_417513,EPI_ISL_417514, EPI_ISL_417515, EPI_ISL_417516, EPI_ISL_417517,EPI_ISL_417526, EPI_ISL_417527, EPI_ISL_417528, EPI_ISL_417529,EPI_ISL_417530, EPI_ISL_417531, EPI_ISL_417532, EPI_ISL_417533,EPI_ISL_417534, EPI_ISL_417536, EPI_ISL_417537, EPI_ISL_417538,EPI_ISL_417539, EPI_ISL_417540, EPI_ISL_417541, EPI_ISL_417542,EPI_ISL_417543, EPI_ISL_417544, EPI_ISL_417545, EPI_ISL_417546,EPI_ISL_417547, EPI_ISL_417548, EPI_ISL_417550, EPI_ISL_417551,EPI_ISL_417552, EPI_ISL_417553, EPI_ISL_417554, EPI_ISL_417555,EPI_ISL_417556, EPI_ISL_417557, EPI_ISL_417558, EPI_ISL_417559,EPI_ISL_417560, EPI_ISL_417561, EPI_ISL_417562, EPI_ISL_417563,EPI_ISL_417564, EPI_ISL_417565, EPI_ISL_417566, EPI_ISL_417567,EPI_ISL_417568, EPI_ISL_417569, EPI_ISL_417570, EPI_ISL_417571,EPI_ISL_417572, EPI_ISL_417573, EPI_ISL_417574, EPI_ISL_417575,EPI_ISL_417576, EPI_ISL_417577, EPI_ISL_417578, EPI_ISL_417579,EPI_ISL_417580, EPI_ISL_417581, EPI_ISL_417582, EPI_ISL_417583,EPI_ISL_417584, EPI_ISL_417585, EPI_ISL_417586, EPI_ISL_417587,EPI_ISL_417588, EPI_ISL_417589, EPI_ISL_417590, EPI_ISL_417591,EPI_ISL_417592, EPI_ISL_417593, EPI_ISL_417594, EPI_ISL_417595,EPI_ISL_417596, EPI_ISL_417597, EPI_ISL_417598, EPI_ISL_417599,EPI_ISL_417600, EPI_ISL_417601, EPI_ISL_417602, EPI_ISL_417603,EPI_ISL_417604, EPI_ISL_417605, EPI_ISL_417606, EPI_ISL_417607,EPI_ISL_417608, EPI_ISL_417609, EPI_ISL_417610, EPI_ISL_417611,EPI_ISL_417612, EPI_ISL_417613, EPI_ISL_417614, EPI_ISL_417615,EPI_ISL_417616, EPI_ISL_417617, EPI_ISL_417618, EPI_ISL_417619,EPI_ISL_417620, EPI_ISL_417621, EPI_ISL_417622, EPI_ISL_417623,EPI_ISL_417624, EPI_ISL_417625, EPI_ISL_417626, EPI_ISL_417627,EPI_ISL_417628, EPI_ISL_417629, EPI_ISL_417630, EPI_ISL_417631,EPI_ISL_417632, EPI_ISL_417633, EPI_ISL_417634, EPI_ISL_417635,EPI_ISL_417636, EPI_ISL_417637, EPI_ISL_417638, EPI_ISL_417639,EPI_ISL_417640, EPI_ISL_417641, EPI_ISL_417642, EPI_ISL_417643,EPI_ISL_417644, EPI_ISL_417645, EPI_ISL_417646, EPI_ISL_417647,EPI_ISL_417648, EPI_ISL_417649, EPI_ISL_417650, EPI_ISL_417651,EPI_ISL_417652, EPI_ISL_417653, EPI_ISL_417654, EPI_ISL_417666,EPI_ISL_417667, EPI_ISL_417668, EPI_ISL_417669, EPI_ISL_417670,EPI_ISL_417671, EPI_ISL_417672, EPI_ISL_417676, EPI_ISL_417678,EPI_ISL_417680, EPI_ISL_417685, EPI_ISL_417699, EPI_ISL_417700,EPI_ISL_417703, EPI_ISL_417706, EPI_ISL_417709, EPI_ISL_417712,EPI_ISL_417716, EPI_ISL_417717, EPI_ISL_417724, EPI_ISL_417733,EPI_ISL_417737, EPI_ISL_417740, EPI_ISL_417742, EPI_ISL_417743,EPI_ISL_417746, EPI_ISL_417750, EPI_ISL_417752, EPI_ISL_417753,EPI_ISL_417754, EPI_ISL_417762, EPI_ISL_417763, EPI_ISL_417764,EPI_ISL_417766, EPI_ISL_417774, EPI_ISL_417808, EPI_ISL_417809,EPI_ISL_417813, EPI_ISL_417814, EPI_ISL_417815, EPI_ISL_417816,EPI_ISL_417818, EPI_ISL_417819, EPI_ISL_417820, EPI_ISL_417821,EPI_ISL_417822, EPI_ISL_417823, EPI_ISL_417824, EPI_ISL_417825,EPI_ISL_417826, EPI_ISL_417827, EPI_ISL_417829, EPI_ISL_417830,EPI_ISL_417831, EPI_ISL_417832, EPI_ISL_417833, EPI_ISL_417834,EPI_ISL_417835, EPI_ISL_417836, EPI_ISL_417837, EPI_ISL_417838,EPI_ISL_417839, EPI_ISL_417864, EPI_ISL_417917, EPI_ISL_417918,EPI_ISL_417920, EPI_ISL_417925, EPI_ISL_417926, EPI_ISL_417931,EPI_ISL_417932, EPI_ISL_417933, EPI_ISL_417935, EPI_ISL_417936,EPI_ISL_417937, EPI_ISL_417938, EPI_ISL_417939, EPI_ISL_417940,EPI_ISL_417941, EPI_ISL_417942, EPI_ISL_417943, EPI_ISL_417944,EPI_ISL_417945, EPI_ISL_417946, EPI_ISL_417947, EPI_ISL_417948,EPI_ISL_417949, EPI_ISL_417950, EPI_ISL_417951, EPI_ISL_417953,EPI_ISL_417955, EPI_ISL_417958, EPI_ISL_417959, EPI_ISL_417960,EPI_ISL_417962, EPI_ISL_417964, EPI_ISL_417965, EPI_ISL_417966,EPI_ISL_417968, EPI_ISL_417970, EPI_ISL_417971, EPI_ISL_417973,EPI_ISL_417974, EPI_ISL_417976, EPI_ISL_417977, EPI_ISL_417982,EPI_ISL_417983, EPI_ISL_417984, EPI_ISL_417985, EPI_ISL_418009,EPI_ISL_418017, EPI_ISL_418018, EPI_ISL_418019, EPI_ISL_418020,EPI_ISL_418021, EPI_ISL_418022, EPI_ISL_418023, EPI_ISL_418024,EPI_ISL_418025, EPI_ISL_418026, EPI_ISL_418027, EPI_ISL_418029,EPI_ISL_418030, EPI_ISL_418031, EPI_ISL_418032, EPI_ISL_418033,EPI_ISL_418034, EPI_ISL_418037, EPI_ISL_418038, EPI_ISL_418040,EPI_ISL_418046, EPI_ISL_418047, EPI_ISL_418048, EPI_ISL_418050,EPI_ISL_418052, EPI_ISL_418053, EPI_ISL_418054, EPI_ISL_418063,EPI_ISL_418064, EPI_ISL_418067, EPI_ISL_418071, EPI_ISL_418072,EPI_ISL_418073, EPI_ISL_418074, EPI_ISL_418075, EPI_ISL_418076,EPI_ISL_418077, EPI_ISL_418078, EPI_ISL_418079, EPI_ISL_418080,EPI_ISL_418081, EPI_ISL_418082, EPI_ISL_418101, EPI_ISL_418102,EPI_ISL_418103, EPI_ISL_418104, EPI_ISL_418105, EPI_ISL_418126,EPI_ISL_418127, EPI_ISL_418128, EPI_ISL_418129, EPI_ISL_418130,EPI_ISL_418131, EPI_ISL_418132, EPI_ISL_418133, EPI_ISL_418134,EPI_ISL_418135, EPI_ISL_418136, EPI_ISL_418137, EPI_ISL_418138,EPI_ISL_418139, EPI_ISL_418140, EPI_ISL_418148, EPI_ISL_418149,EPI_ISL_418150, EPI_ISL_418151, EPI_ISL_418152, EPI_ISL_418153,EPI_ISL_418154, EPI_ISL_418155, EPI_ISL_418156, EPI_ISL_418157,EPI_ISL_418158, EPI_ISL_418159, EPI_ISL_418160, EPI_ISL_418161,EPI_ISL_418162, EPI_ISL_418163, EPI_ISL_418164, EPI_ISL_418165,EPI_ISL_418183, EPI_ISL_418184, EPI_ISL_418185, EPI_ISL_418186,EPI_ISL_418187, EPI_ISL_418188, EPI_ISL_418189, EPI_ISL_418190,EPI_ISL_418191, EPI_ISL_418192, EPI_ISL_418193, EPI_ISL_418194,EPI_ISL_418195, EPI_ISL_418197, EPI_ISL_418198, EPI_ISL_418199,EPI_ISL_418200, EPI_ISL_418201, EPI_ISL_418202, EPI_ISL_418203,EPI_ISL_418204, EPI_ISL_418231, EPI_ISL_418232, EPI_ISL_418233,EPI_ISL_418235, EPI_ISL_418236, EPI_ISL_418237, EPI_ISL_418238,EPI_ISL_418239, EPI_ISL_418240, EPI_ISL_418257, EPI_ISL_418260,EPI_ISL_418263, EPI_ISL_418264, EPI_ISL_418265 or EPI_ISL_616802(hCoV-19/Denmark/DCGC-3024/2020).

Exemplary SARS-CoV-2 coronaviruses can also be defined or identified bygenetic information provided by GenBank Accession Numbers as provided inList B below.

List B: GenBank Accession Numbers of Different SARS-CoV-2 Isolates:

NC_045512, LC528232, LC528233, LC529905, MN908947, MN938384, MN938385,MN938386, MN938387, MN938388, MN938389, MN938390, MN970003, MN970004,MN975262, MN975263, MN975264, MN975265, MN975266, MN975267, MN975268,MN985325, MN988668, MN988669, MN994467, MN994468, MN996527, MN996528,MN996529, MN996530, MN996531, MN997409, MT007544, MT012098, MT019529,MT019530, MT019531, MT019532, MT019533, MT020880, MT020881, MT027062,MT027063, MT027064, MT039873, MT039887, MT039888, MT039890, MT044257,MT044258, MT049951, MT050493, MT066156, MT066175, MT066176, MT072688,MT093571, MT093631, MT106052, MT106053, MT106054, MT118835, MT121215,MT123290, MT123291, MT123292, MT123293, MT126808, MT135041, MT135042,MT135043, MT135044, MT152824, MT159705, MT159706, MT159707, MT159708,MT159709, MT159710, MT159711, MT159712, MT159713, MT159714, MT159715,MT159716, MT159717, MT159718, MT159719, MT159720, MT159721, MT159722,MT163716, MT163717, MT163718, MT163719, MT163720, MT163721, MT184907,MT184908, MT184909, MT184910, MT184911, MT184912, MT184913, MT188339,MT188340, MT188341, MT192759, MT192765, MT192772 or MT192773.

SARS-CoV-2 coronavirus has been attributed the NCBI Taxonomy ID (NCBI:txid or taxID): 2697049.

The term “antigenic peptide or protein of an nCoV-2019 coronavirus” or“antigenic peptide or protein of a SARS-CoV-2 coronavirus” relates toany peptide or protein that is (from) or is derived from a SARS-CoV-2(nCoV-2019) coronavirus as defined above, but also to fragments,variants or derivatives thereof, preferably to immunogenic fragments orimmunogenic variants thereof.

The term “immunogenic fragment” or “immunogenic variant” has to beunderstood as any fragment/variant of the corresponding SARS-CoV-2(nCoV-2019) coronavirus antigen that is capable of raising an immuneresponse in a subject. Preferably, intramuscular or intradermaladministration of the nucleic acid of the first aspect results inexpression of the encoded SARS-CoV-2 antigen (peptide or protein) in asubject.

The term “expression” as used herein refers to the production of aSARS-CoV-2 coronavirus peptide or protein, wherein said SARS-CoV-2coronavirus peptide or protein is provided by a coding sequence of anucleic acid of the first aspect. For example, “expression” of an RNArefers to production of a protein (e.g. after administration of said RNAto a cell or a subject) via translation of the RNA into a polypeptide,e.g. into a peptide or protein that is or is derived from a SARS-CoV-2coronavirus. “Expression” of a DNA refers to production of a protein(e.g. after administration of said DNA to a cell or a subject) viatranscription of the DNA into RNA and subsequent translation into apolypeptide, e.g. into a peptide or protein that is or is derived from aSARS-CoV-2 coronavirus. The term “expression” and the term “production”may be used interchangeably herein. Further, the term “expression”preferably relates to production of a certain peptide or protein uponadministration of a nucleic acid to a cell or an organism. Inembodiments, the nucleic acid is suitable for a vaccine, preferably acoronavirus vaccine. In preferred embodiments, the nucleic acid issuitable for a SARS-CoV-2 coronavirus vaccine.

In the context of the invention, any protein that is or is derived froma SARS-CoV-2 coronavirus may be used and may be suitably encoded by thecoding sequence or the nucleic acid of the first aspect. It is furtherin the scope of the underlying invention, that the at least oneantigenic peptide or protein may comprise or consist of a syntheticallyengineered or an artificial coronavirus peptide or protein. The term“synthetically engineered” coronavirus peptide or protein, or the term“artificial coronavirus peptide or protein” relates to a protein thatdoes not occur in nature. Accordingly, an “artificial coronaviruspeptide or protein” or a “synthetically engineered coronavirus peptideor protein” may for example differ in at least one amino acid comparedto the natural coronavirus peptide or protein, and/or may comprise anadditional heterologous peptide or protein element, and/or may beN-terminally or C-terminally extended or truncated.

In preferred embodiments, the nucleic acid comprises at least one codingsequence encoding at least one antigenic peptide or protein ofSARS-CoV-2 coronavirus, or an immunogenic fragment or immunogenicvariant thereof, wherein the at least one antigenic peptide or proteincomprises at least one peptide or protein that is or is derived from astructural protein, an accessory protein, or a replicase protein or animmunogenic fragment or immunogenic variant of any of these.

In preferred embodiments, the nucleic acid comprises at least one codingsequence encoding at least one antigenic peptide or protein ofSARS-CoV-2 coronavirus, wherein the at least one antigenic peptide orprotein comprises at least one peptide or protein that is or is derivedfrom a structural protein, wherein the structural protein is selectedfrom a spike protein (S), an envelope protein (E), a membrane protein(M) or a nucleocapsid protein (N), or an immunogenic fragment or variantof any of these.

In particularly preferred embodiments, the encoded at least oneantigenic peptide or protein comprises or consists of a spike protein(S), or an immunogenic fragment or immunogenic variant thereof.

Spike protein is a typical type I viral fusion protein that exists astrimer on the viral surface with each monomer consisting of a Head (S1)and stem (S2). Individual precursor S polypeptides form a homotrimer andundergo glycosylation within the Golgi apparatus as well as processingto remove the signal peptide, and cleavage by a cellular protease togenerate separate S1 and S2 polypeptide chains, which remain associatedas S1/S2 protomers within the homotrimer and is therefore a trimer ofheterodimers. The S1 domain of the spike glycoprotein includes thereceptor binding domain (RBD) that engages (most likely) with theangiotensin-converting enzyme 2 receptors and mediates viral fusion intothe host cell, an N-terminal domain that may make initial contact withtarget cells, and 2 subdomains, all of which are susceptible toneutralizing antibodies. S2 domain consists of a six helix bundle fusioncore involved in membrane fusion with the host endosomal membrane and isalso a target for neutralization. The S2 subunit further comprises twoheptad-repeat sequences (HR1 and HR2) and a central helix typical offusion glycoproteins, a transmembrane domain, and the cytosolic taildomain.

Suitable antigenic peptide or protein sequences that are provided by thenucleic acid of the invention are disclosed in Table 1, rows 1 to 41,Column A and B. In addition, further information regarding said suitableantigenic peptide or protein sequences are provided under <223>identifier of the ST25 sequence listing.

In the following, preferred antigenic peptide or protein sequences thatare provided by the nucleic acid of the invention are described indetail.

In preferred embodiments, the encoded at least one antigenic peptide orprotein comprises or consists of at least one of the amino acidsequences being identical or at least 50%, 60%, 70%, 80%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identicalto any one of SEQ ID NOs: 1-111, 274-11663, 13176-13510, 13521-14123,22732-22758, 22917, 22923, 22929-22964, 26938, 26939 or an immunogenicfragment or immunogenic variant of any of these. Further informationregarding said amino acid sequences is also provided in Table 1 (seerows 1 to 41 of Column A and B), and under <223> identifier of the ST25sequence listing of respective sequence SEQ ID NOs.

It has to be noted that where reference is made to amino acid (aa)residues and their position in a spike protein (S), any numbering usedherein—unless stated otherwise—relates to the position of the respectiveamino acid residue in a corresponding spike protein (S) of SARS-CoV-2(nCoV-2019) coronavirus isolate EPI_ISL_402128(BetaCoV_Wuhan_WIV05_2019_EPI_ISL_402128) according to SEQ ID NO: 1.Respective amino acid positions are, throughout the disclosure,exemplarily indicated for spike protein (S) of SARS-CoV-2 coronavirusisolate EPI_ISL_402128 (SEQ ID NO: 1). The person skilled in the artwill of course be able to adapt the teaching provided in the presentspecification exemplified for SARS-CoV-2 EPI_ISL_402128 (SEQ ID NO: 1)to other antigenic peptides or proteins in other SARS-CoV-2 coronavirusisolates, e.g. to isolates including but not limited to EPI_ISL_404227,EPI_ISL_403963, EPI_ISL_403962, EPI_ISL_403931, EPI_ISL_403930,EPI_ISL_403929, EPI_ISL_402130, EPI_ISL_402129, EPI_ISL_402128,EPI_ISL_402126, EPI_ISL_402125, EPI_ISL_402124, EPI_ISL_402123,EPI_ISL_402120, EPI_ISL_402119 (further SARS-CoV-2 isolates are providedin List A and/or List B and or Table 25).

Protein annotation was performed using SEQ ID NO: 1 as a referenceprotein. The full-length spike protein (S) of SARS-CoV-2 coronavirusreference protein has 1273 amino acid residues, and comprises thefollowing elements:

-   -   secretory signal peptide: amino acid position aa 1 to aa 15 (see        SEQ ID NO: 28)    -   spike protein fragment S1: amino acid position aa 1 to aa 681        (see SEQ ID NO: 27)    -   receptor binding domain (RBD): amino acid position aa 319 to aa        541 (see SEQ ID NO: 13243)    -   critical neutralisation domain (CND): amino acid position aa 329        to aa 529 (see SEQ ID NO: 13310)    -   spike protein fragment S2: amino acid position aa 682 to aa 1273        (see SEQ ID NO: 30)    -   transmembrane domain TM amino acid position aa 1212 to aa 1273        (see SEQ ID NO: 49)    -   transmembrane domain (TMflex) amino acid position aa 1148 to aa        1273 (see SEQ ID NO: 13176)

It has to be noted that variation on amino acid level naturally occursbetween spike proteins derived from different SARS-CoV-2 isolates(exemplary SARS-CoV-2 isolates are provided in List A and List B). Inthe context of the invention, such amino acid variations can be appliedto each antigenic peptide or protein derived from a spike protein asdescribed herein.

Accordingly, each spike protein provided herein and contemplated assuitable antigen in the context of the invention may have one or more ofthe following amino acid variations (amino acid positions according toreference SEQ ID NO: 1):

-   -   D614G or G614D    -   H49Y or Y49H    -   V367F or F367V    -   P1263L or L1263P    -   V483A or A483V    -   S939F or F939S    -   S943P or P943S    -   L5F or F5L    -   L8V or V8L    -   S940F or F940S    -   C1254F or F1254C    -   Q239K or K239Q    -   M153T or T153M    -   V1040F or F1040V    -   A845S or S845A    -   Y145H or H145Y    -   A831V or V831A    -   M1229I or I1229M    -   H69 or H69del/aa deleted    -   V70 or H70del/aa deleted    -   H69_V70 or H69del and H70del/aa deleted    -   A222V or V222A    -   Y453F or F453Y    -   S477N or N477S    -   I692V or V692I    -   R403K or K403R    -   K417N or N417K    -   N437S or S437N    -   N439K or K439N    -   V445A or A445V    -   V445I or I445V    -   V445F or F445V    -   G446V or V446G    -   G446S or S446G    -   G446A or A446G    -   L455F or F455L    -   F456L or L456F    -   K458N or N458K    -   A475V or V475A    -   G476S or S476G    -   G476A or A476G    -   S4771 or I477S    -   S477R or R477S    -   S477G or G477S    -   S477T or T477S    -   T4781 or I478T    -   T478K or K478T    -   T478R or R478T    -   T478A or A478T    -   E484Q or Q484E    -   E484K or K484E    -   E484A or A484E    -   E484D or D484E    -   G485R or R485G    -   G485S or S485G    -   F486L or L486F    -   N4871 or I487N    -   Y489H or H489Y    -   F490S or S490F    -   F490L or L490F    -   Q493L or L493Q    -   Q493K or K493Q    -   S494P or P494S    -   S494L or L494S    -   P499L or L499P    -   T5001 or 1500T    -   N501Y or Y501N    -   N501T or T501N    -   N501S or S501N    -   V503F or F503V    -   V5031 or 1503V    -   G504D or D504G    -   Y505W or W505Y    -   Q506K or K506Q    -   0506H or H5060    -   Y144 or Y144del/aa deleted    -   A570D or D570A    -   P681H or H681P    -   T716I or I716T    -   S982A or A982S    -   D1118H or H1118D    -   L18F or F18L    -   D80A or A80D    -   D215G or G215D    -   L242 or L242del/aa deleted    -   A243 or A243del/aa deleted    -   L244 or L244del/aa deleted    -   L242_A243_L244 or L242del and A243del and L244del/aa deleted    -   R2461 or 1246R    -   A701V or V701A    -   T20N or N20T    -   P26S or S26P    -   D138Y or Y138D    -   R190S or S190R    -   H655Y or Y655H    -   T10271 or I1027T    -   S13I or I13S    -   W152C or C152W    -   L452R or R452L    -   R346T or T346R    -   P384L or L384P    -   L452M or M452L    -   F456A or A456F    -   F456K or K456F    -   F456V or V456F    -   E484P or P484E    -   K417T or T417K    -   G447V or V447G    -   L452Q or Q452L    -   A475S or S475A    -   F4861 or 1486F    -   F490Y or Y490F    -   Q493R or R493Q    -   S494A or A494S    -   P499H or H499P    -   P499S or S499P    -   G502V or V502G    -   T748K or K748T    -   A522S or S522A    -   V1176F or F1176V

The following amino acid variations (amino acid positions according toreference SEQ ID NO: 1) are particularly preferred:

-   -   H69del, V70del, Y144del, N501Y, A570D, D614G, P681 H, T716I,        S982A, and D1118H    -   L18F, D80A, D215G, L242del, A243del, L244del, R2461, K417N,        E484K, N501Y, D614G, and A701V    -   K417N, E484K, N501Y, and D614G    -   E484K and D614G    -   L18F, T20N, P26S, D138Y, R190S, K417T, E484K, N501Y, D614G,        H655Y, and T10271    -   S13I, W152C, L452R, and D614G    -   delH69, delV70, Y453F, D614G, 1692V, and M1229I    -   E484K, E484P, or E484Q    -   G446V    -   G485R

In some embodiments, a fragment of a spike protein (S) may be encoded bythe nucleic acid of the invention, wherein said fragment may beN-terminally truncated, lacking the N-terminal amino acids 1 to up to100 of the full length SARS-CoV-2 coronavirus reference protein (SEQ IDNO: 1) and/or wherein said fragment may be C-terminally truncated,lacking the C-terminal amino acids (aa) 531 to up to aa 1273 of the fulllength SARS-CoV-2 coronavirus reference protein (SEQ ID NO: 1). Such“fragment of a spike protein (S)” may additionally comprise amino acidsubstitutions (as described below) and may additionally comprise atleast one heterologous peptide or protein element (as described below).In preferred embodiments, a fragment of a spike protein (S) may beC-terminally truncated, thereby lacking the C-terminal transmembranedomain (that is, lacking aa 1212 to aa 1273 or lacking aa 1148 to aa1273).

In other embodiments, the encoded at least one antigenic peptide orprotein comprises or consists of a spike protein (S), wherein the spikeprotein (S) derived from SARS-CoV-2 coronavirus lacks the transmembranedomain TM (amino acid position aa 1212 to aa 1273). In embodiments, theencoded at least one antigenic peptide or protein comprises or consistsof a spike protein (S), wherein the spike protein (S) derived fromSARS-CoV-2 coronavirus lacks an extended part of the transmembranedomain (TMflex) (amino acid position aa 1148 to aa 1273). Withoutwishing to being bound to theory, a spike protein (S) lacking thetransmembrane domain (TM or TMflex) as defined herein could be suitablefor a coronavirus vaccine, as such a protein would be soluble and notanchored in the cell membrane. A soluble protein may therefore beproduced (that is translated) in higher concentrations uponadministration to a subject, leading to improved immune responses.

Without wishing to being bound to theory, RBD (aa 319 to aa 541) and CND(aa 29 to aa 529) domains may be crucial for immunogenicity. Bothregions are located at the S1 fragment of the spike protein.Accordingly, it may be suitable in the context of the invention that theantigenic peptide or protein comprises or consists of an S1 fragment ofthe spike protein or an immunogenic fragment or immunogenic variantthereof. Suitably, such an S1 fragment may comprise at least an RBDand/or a CND domain as defined above.

In preferred embodiments, the encoded at least one antigenic peptide orprotein comprises or consists of a receptor-binding domain (RBD; aa 319to aa 541), wherein the RBD comprises or consists of a spike proteinfragment, or an immunogenic fragment or immunogenic variant thereof.

In further preferred embodiments, the encoded at least one antigenicpeptide or protein comprises or consists of a truncated receptor-bindingdomain (truncRBD; aa 334 to aa 528), wherein the RBD comprises orconsists of a spike protein fragment, or an immunogenic fragment orimmunogenic variant thereof.

Such “fragment of a spike protein (S)” (RBD; aa 319 to aa 541 ortruncRBD, aa 334 to aa 528), may additionally comprise amino acidsubstitutions (as described below) and may additionally comprise atleast one heterologous peptide or protein element (as described below).

In particularly preferred embodiments, the encoded at least oneantigenic peptide or protein comprises or consists of a spike protein(S), wherein the spike protein (S) comprises or consists of a spikeprotein fragment S1, or an immunogenic fragment or immunogenic variantthereof.

Accordingly, in preferred embodiments, the encoded at least oneantigenic peptide or protein (comprising or consisting of a spikeprotein fragment S1) comprises or consists of at least one of the aminoacid sequences being identical or at least 50%, 60%, 70%, 80%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to any one of SEQ ID NOs: 1-27, 29, 31-48, 58-111, 274-1345,1480-1546, 1614-11663, 13377-13510, 13521-14123, 22732, 22737-22758,22929-22964 or an immunogenic fragment or immunogenic variant of any ofthese. Further information regarding said amino acid sequences is alsoprovided in Table 1 (see rows 1 to 6, 9, 11-41 of Column A and B), andunder <223> identifier of the ST25 sequence listing of respectivesequence SEQ ID NOs.

In preferred embodiments, the encoded at least one antigenic peptide orprotein comprises an spike protein fragment S1, and lacks at least 70%,80%, 90%, preferably 100% of spike protein fragment S2 (aa 682 to aa1273). Such embodiments may be beneficial, as the S1 fragment comprisesneutralizing epitopes without potential problems of full-length proteincomprising S1 and S2.

Accordingly, in preferred embodiments, the encoded at least oneantigenic peptide or protein (essentially consisting of a spike proteinfragment S1) comprises or consists of at least one of the amino acidsequences being identical or at least 50%, 60%, 70%, 80%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identicalto any one of SEQ ID NOs: 27, 1279-1345, 29, 1480-1546, 13243-13309,22733-22736, 26938, 26939 or an immunogenic fragment or immunogenicvariant of any of these. Further information regarding said amino acidsequences is also provided in Table 1 (see rows 6 and 9 of Column A andB), and under <223> identifier of the ST25 sequence listing ofrespective sequence SEQ ID NOs.

Without wishing to being bound to theory, it may be suitable that theantigenic peptide or protein comprises or consists of spike proteinfragment S1 and (at least a fragment of) spike protein fragment S2,because the formation of an immunogenic spike protein may be promoted.

Accordingly, in particularly preferred embodiments, the encoded at leastone antigenic peptide or protein comprises or consists of a spikeprotein (S), wherein the spike protein (S) comprises or consists of aspike protein fragment S1 or an immunogenic fragment or immunogenicvariant thereof, and spike protein fragment S2 or an immunogenicfragment or immunogenic variant thereof.

In preferred embodiments, the at least one encoded antigenic peptide orprotein that comprises or consists of spike protein fragment S1 andspike protein fragment S2, comprises at least one of the amino acidsequences being identical or at least 50%, 60%, 70%, 80%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identicalto any one of SEQ ID NOs: 1-26, 31-48, 58-111, 274-1278, 1614-11663,13377-13510, 13521-14177, 22732, 22737-22758, 22929-22964 or animmunogenic fragment or immunogenic variant of any of these. Furtherinformation regarding said amino acid sequences is also provided inTable 1 (see rows 1 to 5, 11-35, 38 of Column A and B), and under <223>identifier of the ST25 sequence listing of respective sequence SEQ IDNOs.

In particularly preferred embodiments, the encoded at least oneantigenic peptide or protein comprises or consists of a full-lengthspike protein or an immunogenic fragment or immunogenic variant of anyof these.

The term “full length spike protein” has to be understood as a spikeprotein, preferably derived from a SARS-CoV-2 coronavirus, having anamino acid sequence corresponding to essentially the full spike protein.Accordingly, a “full length spike protein” may comprise aa 1 to aa 1273(reference protein: SEQ ID NOs: 1). Accordingly, a full length spikeprotein may typically comprise a secretory signal peptide, a spikeprotein fragment S1, a spike protein fragment S2, a receptor bindingdomain (RBD), and a critical neutralisation domain CND, and atransmembrane domain. Notably, also variants that comprise certain aminoacid substitutions (e.g. for allowing pre-fusion stabilization of the Sprotein) or natural occurring amino acid deletions are encompassed bythe term “full length spike protein”.

Accordingly, in preferred embodiments, the at least one encodedantigenic peptide or protein is a full length S protein comprising orconsisting of at least one of the amino acid sequences being identicalor at least 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs:1-9, 274-340, 22737, 22739, 22741, 22743, 22745, 22747, 22749, 22751,22753, 22755, 22757, 22929-22946 or an immunogenic fragment orimmunogenic variant of any of these. Further information regarding saidamino acid sequences is also provided in Table 1 (see row 1 of Column Aand B), and under <223> identifier of the ST25 sequence listing ofrespective sequence SEQ ID NOs.

In particularly preferred embodiments, the spike protein (S) that isprovided by the nucleic acid of the first aspect is designed or adaptedto stabilize the antigen in pre-fusion conformation. A pre-fusionconformation is particularly advantageous in the context of an efficientcoronavirus vaccine, as several potential epitopes for neutralizingantibodies may merely be accessible in said pre-fusion proteinconformation. Furthermore, remaining of the protein in the pre-fusionconformation is aimed to avoid immunopathological effects, like e.g.enhanced disease and/or antibody dependent enhancement (ADE).

In preferred embodiments, administration of a nucleic acid (or acomposition or vaccine) encoding pre-fusion stabilized spike protein toa subject elicits spike protein neutralizing antibodies and does notelicit disease-enhancing antibodies. In particular, administration of anucleic acid (or a composition or vaccine) encoding pre-fusionstabilized spike protein to a subject does not elicit immunopathologicaleffects, like e.g. enhanced disease and/or antibody dependentenhancement (ADE).

Accordingly, in preferred embodiments, the nucleic acid of the inventioncomprises at least one coding sequence encoding at least one antigenicpeptide or protein that is or is derived from an SARS-CoV-2 coronavirus,wherein the at least one antigenic peptide or protein is or is derivedfrom a spike protein (S), wherein the spike protein (S) is a pre-fusionstabilized spike protein (S_stab). Suitably, said pre-fusion stabilizedspike protein comprises at least one pre-fusion stabilizing mutation.

The term “pre-fusion conformation” as used herein relates to astructural conformation adopted by the ectodomain of the coronavirus Sprotein following processing into a mature coronavirus S protein in thesecretory system, and prior to triggering of the fusogenic event thatleads to transition of coronavirus S to the postfusion conformation.

A “pre-fusion stabilized spike protein (S_stab)” as described hereincomprises one or more amino acid substitutions, deletions, or insertionscompared to a native coronavirus S sequence that provide for increasedretention of the prefusion conformation compared to coronavirus Sectodomain trimers formed from a corresponding native coronavirus Ssequence. The “stabilization” of the prefusion conformation by the oneor more amino acid substitutions, deletions, or insertions can be, forexample, energetic stabilization (for example, reducing the energy ofthe prefusion conformation relative to the post-fusion openconformation) and/or kinetic stabilization (for example, reducing therate of transition from the prefusion conformation to the postfusionconformation). Additionally, stabilization of the coronavirus Sectodomain trimer in the prefusion conformation can include an increasein resistance to denaturation compared to a corresponding nativecoronavirus S sequence.

Accordingly, in preferred embodiments, the spike protein includes one ormore amino acid substitutions that stabilize the S protein in thepre-fusion conformation, for example, substitutions that stabilize themembrane distal portion of the S protein (including the N-terminalregion) in the pre-fusion conformation.

Stabilization of the SARS-CoV-2 coronavirus spike protein may beobtained by substituting at least one amino acid at position K986 and/orV987 with amino acids that stabilize the spike protein in a perfusionconformation (amino acid positions according to reference SEQ ID NO: 1).

In particularly preferred embodiments, the pre-fusion stabilizingmutation comprises an amino acid substitution at position K986, whereinthe amino acids K986 is substituted with one selected from A, I, L, M,F, V, G, or P (amino acid positions according to reference SEQ ID NO:1), preferably wherein the amino acids K986 is substituted with P. Inadditionally preferred embodiments, the pre-fusion stabilizing mutationcomprises an amino acid substitution at position K986, wherein the aminoacids V987 is substituted with one selected from A, I, L, M, F, V, G, orP (amino acid positions according to reference SEQ ID NO: 1), preferablywherein the amino acids V987 is substituted with P.

Suitably, stabilization of the SARS-CoV-2 coronavirus spike protein maybe obtained by substituting two consecutive amino acids at position K986and V987 with amino acids that stabilize the spike protein in aprefusion conformation (Amino acid positions according to reference SEQID NO: 1).

In preferred embodiments, the pre-fusion stabilizing mutation comprisesan amino acid substitution at position K986 and V987, wherein the aminoacids K986 and/or V987 are substituted with one selected from A, I, L,M, F, V, G, or P (amino acid positions according to reference SEQ ID NO:1).

Preferably, stabilization of the perfusion conformation is obtained byintroducing two consecutive proline substitutions at residues K986 andV987 in the spike protein (Amino acid positions according to referenceSEQ ID NO: 1).

Accordingly, in preferred embodiments, the pre-fusion stabilized spikeprotein (S_stab) comprises at least one pre-fusion stabilizing mutation,wherein the at least one pre-fusion stabilizing mutation comprises thefollowing amino acid substitutions: K986P and V987P (amino acidpositions according to reference SEQ ID NO: 1).

Accordingly, any NCBI Protein Accession numbers provided above, or anyprotein selected from SEQ ID NOs: 1-9, 274-340, 22737, 22739, 22741,22743, 22745, 22747, 22749, 22751, 22753, 22755, 22757, 22929-22946 orfragments or variants thereof can be chosen by the skilled person tointroduce such amino acid changes, preferably amino acid substitutions:K986P and V987P (amino acid positions according to reference SEQ ID NO:1).

In preferred embodiments, the at least one pre-fusion stabilizingmutation comprises a cavity filling mutation that further stabilizes thepre-fusion state, wherein said mutation/amino acid substitution isselected from the list comprising T887WN; A1020W; T887WN and A1020W; orP1069F (amino acid positions according to reference SEQ ID NO: 1).

The term “cavity filling mutation” or “cavity filling amino acidsubstitution” relates to an amino acid substitution that fills a cavitywithin the protein core of a protein, such as a coronavirus S proteinectodomain. Cavities are essentially voids within a folded protein whereamino acids or amino acid side chains are not present. In severalembodiments, a cavity-filling amino acid substitution is introduced tofill a cavity present in the prefusion conformation of a coronavirus Sectodomain core that collapses (e.g., has reduced volume) aftertransition to the postfusion conformation.

In some embodiments, at least one of the following amino acidsubstitutions F817P, A892P, A899P and A942P may be combined with a(K986P and V987P) substitution (amino acid positions according toreference SEQ ID NO: 1).

In preferred embodiments, the SARS-CoV-2 coronavirus spike proteincomprises at least one of the following amino acid substitutions (Aminoacid positions according to reference SEQ ID NO: 1):

-   -   F817P; K986P and V987P    -   A892P; K986P and V987P    -   A899P; K986P and V987P    -   A942P; K986P and V987P

In particularly preferred embodiments, the SARS-CoV-2 coronavirus spikeprotein comprises the following amino acid substitutions (Amino acidpositions according to reference SEQ ID NO: 1):

-   -   F817P, A892P, A899P, A942P, K986P and V987P (S_stab_PP_hex)

Accordingly, any NCBI protein accession numbers provided above, or anyprotein selected from SEQ ID NOs: 1-9, 274-340, 22737, 22739, 22741,22743, 22745, 22747, 22749, 22751, 22753, 22755, 22757, 22929-22946 orfragments or variants thereof can be chosen by the skilled person tointroduce such amino acid changes, suitably amino acid substitutionsselected from F817P, A892P, A899P, A942P; or amino acid substitutionsselected from (F817P; K986P and V987P); (A892P; K986P and V987P);(A899P; K986P and V987P); (A942P; K986P and V987P); (F817P, A892P,A899P, A942P, K986P and V987P) (amino acid positions according toreference SEQ ID NO: 1).

In particularly preferred embodiments, at least one of the followingamino acid substitutions T887W; A1020W; T887W and A1020W; or P1069F maybe combined with a (K986P and V987P) substitution (amino acid positionsaccording to reference SEQ ID NO: 1).

In other particularly preferred embodiments, the SARS-CoV-2 coronavirusspike protein comprises at least one of the following amino acidsubstitutions (Amino acid positions according to reference SEQ ID NO:1):

-   -   T887W; K986P and V987P    -   A1020W; K986P and V987P    -   T887W and A1020W; K986P and V987P    -   P1069F; K986P and V987P

Accordingly, any NCBI protein accession numbers provided above, or anyprotein selected from SEQ ID NOs: 1-9, 274-340, 22737, 22739, 22741,22743, 22745, 22747, 22749, 22751, 22753, 22755, 22757, 22929-22946 orfragments or variants thereof can be chosen by the skilled person tointroduce such amino acid changes, suitably amino acid substitutionsselected from T887W; A1020W; T887W and A1020W; or P1069F; or amino acidsubstitutions selected from (T887W; K986P and V987P); (A1020W; K986P andV987P); (T887W and A1020W; K986P and V987P); (P1069F; K986P and V987P)(amino acid positions according to reference SEQ ID NO: 1).

In preferred embodiments, the at least one pre-fusion stabilizingmutation comprises a mutated protonation site that further stabilizesthe pre-fusion state, wherein said mutation/amino acid substitution isselected from H1048Q and H1064N; H1083N and H1101N; or H1048Q and H1064Nand H1083N and H1101N (amino acid positions according to reference SEQID NO: 1).

In some embodiments, at least one of the following amino acidsubstitutions H1048Q and H1064N; H1083N and H1101N; or H1048Q and H1064Nand H1083N and H1101N may be combined with a (K986P and V987P)substitution (amino acid positions according to reference SEQ ID NO: 1).

In particularly preferred embodiments, the SARS-CoV-2 coronavirus spikeprotein comprises at least one of the following amino acid substitutions(Amino acid positions according to reference SEQ ID NO: 1):

-   -   H1048Q and H1064N; K986P and V987P    -   H1083N and H1101N; K986P and V987P    -   H1048Q and H1064N and H1083N and H1101N; K986P and V987P

Accordingly, any NCBI protein accession numbers provided above, or anyprotein selected from SEQ ID NOs: 1-9, 274-340, 22737, 22739, 22741,22743, 22745, 22747, 22749, 22751, 22753, 22755, 22757, 22929-22946 orfragments or variants thereof can be chosen by the skilled person tointroduce such amino acid changes, suitably amino acid substitutionsselected from H1048Q and H1064N; H1083N and H1101N; or H1048Q and H1064Nand H1083N and H1101N; or amino acid substitutions selected from (H1048Qand H1064N; K986P and V987P); (H1083N and H1101N; K986P and V987P);(H1048Q and H1064N and H1083N and H1101N; K986P and V987P); (amino acidpositions according to reference SEQ ID NO: 1).

In preferred embodiments, the at least one pre-fusion stabilizingmutation comprises an artificial intramolecular disulfide bond. Such anartificial intramolecular disulfide bond can be introduced to furtherstabilize the membrane distal portion of the S protein (including theN-terminal region) in the pre-fusion conformation; that is, in aconformation that specifically binds to one or more pre-fusionspecification antibodies, and/or presents a suitable antigenic site thatis present on the pre-fusion conformation but not in the post fusionconformation of the S protein.

In preferred embodiments, the at least one pre-fusion stabilizingmutation generates an artificial intramolecular disulfide bond, whereinthe at least one artificial intramolecular disulfide bond is generatedby at least two of the following amino acid substitutions selected fromthe list comprising 17120,17140, P715C, T874C, G889C, A890C, 19090,N914C, Q965C, F970C, A972C, R995C, G999C, S1003C, L1034C, V1040C,Y1047C, S1055C, P1069C, T1077C, or Y1110C, S1123C (amino acid positionsaccording to reference SEQ ID NO: 1).

In preferred embodiments, the at least one pre-fusion stabilizingmutation generates an artificial intramolecular disulfide bond, whereinthe at least one artificial intramolecular disulfide bond is generatedby at least one of the following amino acid substitutions: 17120 andT1077C; 17140 and Y1110C; P715C and P1069C; G889C and L1034C; 19090 andY1047C; Q965C and S1003C; F970C and G999C; A972C and R995C; A890C andV1040C; T874C and S1055C, or N914C and S1123C (amino acid positionsaccording to reference SEQ ID NO: 1).

In further embodiments, the at least one pre-fusion stabilizing mutationcomprises 2, 3, 4, 5, 6, 7, or 8 different artificial intramoleculardisulfide bonds, wherein each may be selected from the following aminoacid substitutions: 17120 and T1077C; 17140 and Y1110C; P715C andP1069C; G889C and L1034C; 19090 and Y1047C; Q965C and S1003C; F970C andG999C; A972C and R995C; A890C and V1040C; T874C and S1055C, or N914C andS1123C (amino acid positions according to reference SEQ ID NO: 1).

In additional embodiments, at least one, preferably 2, 3, 4, 5 or moreof the following amino acid substitutions 17120 and T1077C; 17140 andY1110C; P715C and P1069C; G889C and L1034C; 19090 and Y1047C; Q965C andS1003C; F970C and G999C; A972C and R995C; A890C and V1040C; T874C andS1055C, or N914C and S1123C may be combined with a (K986P and V987P)substitution. For example, a pre-fusion stabilized S protein maycomprise two different artificial intramolecular disulfide bonds, e.g.17120 and T1077C; P715C and P1069C; and additionally a K986P and V987Psubstitution, etc. (amino acid positions according to reference SEQ IDNO: 1).

In particularly preferred embodiments, the SARS-CoV-2 coronavirus spikeprotein comprises at least one of the following amino acid substitutions(amino acid positions according to reference SEQ ID NO: 1):

-   -   I7120 and T1077C; K986P and V987P    -   I7140 and Y1110C; K986P and V987P    -   P715C and P1069C; K986P and V987P    -   G889C and L1034C; K986P and V987P    -   19090 and Y1047C; K986P and V987P    -   Q965C and S1003C; K986P and V987P    -   F970C and G999C; K986P and V987P    -   A972C and R995C; K986P and V987P    -   A890C and V1040C; K986P and V987P    -   T874C and S1055C; K986P and V987P    -   N914C and S1123C; K986P and V987P

Accordingly, any NCBI protein accession numbers provided above, or anyprotein selected from SEQ ID NOs: 1-9, 274-340, 22737, 22739, 22741,22743, 22745, 22747, 22749, 22751, 22753, 22755, 22757, 22929-22946 orfragments or variants thereof can be chosen by the skilled person tointroduce such amino acid changes, suitably amino acid substitutionsselected from 17120 and T1077C; 17140 and Y1110C; P715C and P1069C;G889C and L1034C; 19090 and Y1047C; Q965C and S1003C; F970C and G999C;A972C and R995C; A890C and V1040C; T874C and S1055C, or N914C andS1123C; or amino acid substitutions selected from (1712C; T1077C; K986P;V987P) or (1714C; Y1110C; K986P; V987P) or (P715C; P1069C; K986P; V987P)or (G889C; L1034C; K986P; V987P) or (1909C; Y1047C; K986P; V987P) or(Q965C; S1003C; K986P; V987P) or (F970C; G999C; K986P; V987P) or (A972C;R995C; K986P; V987P) or (A890C and V1040C; K986P and V987P) or (T874Cand S1055C; K986P and V987P) or (N914C and S1123C; K986P and V987P)(amino acid positions according to reference SEQ ID NO: 1).

It has to be emphasized that in the context of the invention anySARS-CoV-2 coronavirus spike protein may be mutated as described above(exemplified for reference protein SEQ ID NO: 1) to stabilize the spikeprotein in the pre-fusion conformation.

Accordingly, in preferred embodiments, the pre-fusion stabilized spikeprotein (S_stab) comprises or consists of at least one of the amino acidsequences being identical or at least 50%, 60%, 70%, 80%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identicalto any one of SEQ ID NOs: 10-26, 40-48, 85-111, 341-1278, 1681-2618,2686-3623, 3691-4628, 4696-5633, 5701-6638, 6706-7643, 7711-8648,8716-9653, 9721-10658, 10726-11663, 13377-13510, 13521-14123, 22732,22738, 22740, 22742, 22744, 22746, 22748, 22750, 22752, 22754, 22756,22758, 22947-22964 or an immunogenic fragment or immunogenic variant ofany of these. Further information regarding said amino acid sequences isalso provided in Table 1 (see rows 2 to 5, 12-15, 17-20, 22-25, 27-30,32-35, 38 of Column A and B), and under <223> identifier of the ST25sequence listing of respective sequence SEQ ID NOs.

In particularly preferred embodiments, the pre-fusion stabilized spikeprotein (S_stab) comprises or consists of at least one of the amino acidsequences being identical or at least 50%, 60%, 70%, 80%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identicalto any one of SEQ ID NOs: 10-26, 341-407, 609-1278, 13521-13587, 22738,22740, 22742, 22744, 22746, 22748, 22750, 22752, 22754, 22756, 22758,22947-22964 or an immunogenic fragment or immunogenic variant of any ofthese. Further information regarding said amino acid sequences is alsoprovided in Table 1 (see rows 2 and 5 of Column A and B), and under<223> identifier of the ST25 sequence listing of respective sequence SEQID NOs.

In a more preferred embodiment, the pre-fusion stabilized spike protein(S_stab) comprises or consists of at least one of the amino acidsequences being identical or at least 50%, 60%, 70%, 80%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identicalto any one of SEQ ID NOs: 10-18, 341-407, 22947-22964 or an immunogenicfragment or immunogenic variant of any of these. Further informationregarding said amino acid sequences is also provided in Table 1 (see row2 of Column A and B), and under <223> identifier of the ST25 sequencelisting of respective sequence SEQ ID NOs.

In a further, more preferred embodiment, the pre-fusion stabilized spikeprotein (S_stab) comprises or consists of at least one of the amino acidsequences being identical or at least 50%, 60%, 70%, 80%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identicalto any one of SEQ ID NOs: 22960, 22961, 22963 or an immunogenic fragmentor immunogenic variant of any of these.

In an even more preferred embodiment, the pre-fusion stabilized spikeprotein (S_stab) comprises or consists of at least one of the amino acidsequences being identical or at least 50%, 60%, 70%, 80%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identicalto any one of SEQ ID NOs: 10 or 341 or an immunogenic fragment orimmunogenic variant of any of these.

According to various preferred embodiments, the nucleic acid of theinvention encodes at least one antigenic peptide or protein fromSARS-CoV-2 coronavirus as defined herein and, additionally, at least oneheterologous peptide or protein element.

Suitably, the at least one heterologous peptide or protein element maypromote or improve secretion of the encoded antigenic peptide or proteinof the invention (e.g. via secretory signal sequences), promote orimprove anchoring of the encoded antigenic peptide or protein of theinvention in the plasma membrane (e.g. via transmembrane elements),promote or improve formation of antigen complexes (e.g. viamultimerization domains or antigen clustering elements), or promote orimprove virus-like particle formation (VLP forming sequence). Inaddition, the nucleic acid of the first aspect may additionally encodepeptide linker elements, self-cleaving peptides, immunologic adjuvantsequences or dendritic cell targeting sequences.

Suitable multimerization domains may be selected from the list of aminoacid sequences according to SEQ ID NOs: 1116-1167 of WO2017/081082, orfragments or variants of these sequences. Suitable transmembraneelements may be selected from the list of amino acid sequences accordingto SEQ ID NOs: 1228-1343 of WO2017/081082, or fragments or variants ofthese sequences. Suitable VLP forming sequences may be selected from thelist of amino acid sequences according to SEQ ID NOs: 1168-1227 of thepatent application WO2017/081082, or fragments or variants of thesesequences. Suitable peptide linkers may be selected from the list ofamino acid sequences according to SEQ ID NOs: 1509-1565 of the patentapplication WO2017/081082, or fragments or variants of these sequences.Suitable self-cleaving peptides may be selected from the list of aminoacid sequences according to SEQ ID NOs: 1434-1508 of the patentapplication WO2017/081082, or fragments or variants of these sequences.Suitable immunologic adjuvant sequences may be selected from the list ofamino acid sequences according to SEQ ID NOs: 1360-1421 of the patentapplication WO2017/081082, or fragments or variants of these sequences.Suitable dendritic cell (DCs) targeting sequences may be selected fromthe list of amino acid sequences according to SEQ ID NOs: 1344-1359 ofthe patent application WO2017/081082, or fragments or variants of thesesequences. Suitable secretory signal peptides may be selected from thelist of amino acid sequences according to SEQ ID NOs: 1-1115 and SEQ IDNO: 1728 of published PCT patent application WO2017/081082, or fragmentsor variants of these sequences

In preferred embodiments, the at least one coding sequence additionallyencodes one or more heterologous peptide or protein elements selectedfrom a signal peptide, a linker peptide, a helper epitope, an antigenclustering element, a trimerization or multimerization element, atransmembrane element, or a VLP forming sequence.

In preferred embodiments, the nucleic acid of the invention encoding atleast one antigenic protein derived from an SARS-CoV-2 coronavirus,additionally encodes at least one heterologous trimerization element, anantigen clustering element, or a VLP forming sequence.

Antigen Clustering Elements or Multimerization Elements

In preferred embodiments, the antigen clustering elements may beselected from a ferritin element, or a lumazine synthase element,surface antigen of Hepatitis B virus (HBsAg), or encapsulin. Expressinga stably clustered spike protein, preferably in in its prefusionconformation may increases the magnitude and breadth of neutralizingactivity against SARS-CoV-2.

Lumazine synthase (Lumazine, LS, LumSynth) is an enzyme withparticle-forming properties, present in a broad variety of organisms andinvolved in riboflavin biosynthesis.

In particularly preferred embodiments, lumazine synthase is used topromote antigen clustering and may therefore promote or enhance immuneresponses of the encoded coronavirus antigen of the invention.

In particularly preferred embodiments, the antigen clustering element(multimerization element) is or is derived from lumazine synthase,wherein the amino acid sequences of said antigen clustering domain ispreferably identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one ofamino acid sequences SEQ ID NO: 112, a fragment or variant thereof.

Ferritin is a protein whose main function is intracellular iron storage.Almost all living organisms produce ferritin which is made of 24subunits, each composed of a four-alpha-helix bundle, that self-assemblein a quaternary structure with octahedral symmetry. Its properties toself-assemble into nanoparticles are well-suited to carry and exposeantigens.

In particularly preferred embodiments, ferritin is used to promote theantigen clustering and may therefore promote immune responses of theencoded coronavirus antigen, preferably spike protein.

In particularly preferred embodiments, the antigen clustering element(multimerization element) is or is derived from ferritin wherein theamino acid sequences of said antigen clustering domain is preferablyidentical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of amino acidsequence SEQ ID NO: 113, a fragment or variant thereof.

In some embodiments, the antigen-clustering domain is a Hepatitis Bsurface antigen (HBsAg). HBsAg forms spherical particles. The additionof a fragment of the surface antigen of Hepatitis B virus (HBsAg)sequence may be particularly effective in enhancing the immune responseof the nucleic-acid-based vaccine against coronavirus.

In particularly preferred embodiments, HBsAg is used to promote theantigen clustering and may therefore promote immune responses of theencoded coronavirus antigen, preferably a spike protein as definedherein.

In some embodiments, the antigen-clustering element is an encapsulinelement. The addition of an encapsulin sequence may be particularlyeffective in enhancing the immune response of the nucleic-acid-basedvaccine against coronavirus. In particularly preferred embodiments,encapsulin is used to promote the antigen clustering and may thereforepromote immune responses of the encoded coronavirus antigen, preferablya spike protein as defined herein.

Encapsulin is a protein isolated from thermophile Thermotoga maritimaand may be used as an element to allow self-assembly of antigens to formantigen (nano)particles. Encapsulin is assembled from 60 copies ofidentical 31 kDa monomers having a thin and icosahedral T=1 symmetriccage structure with interior and exterior diameters of 20 and 24 nm,respectively.

In some embodiments where the coding sequence of the nucleic acidadditionally encodes heterologous antigen clustering element, it isparticularly preferred and suitable to generate a fusion proteincomprising an antigen-clustering element and an antigenic peptide orprotein derived from SARS-CoV-2. Suitably, said antigenic peptide orprotein, preferably the spike protein, is lacking the C-terminaltransmembrane domain TM (lacking aa 1212 to aa 1273) or is lacking apart of the C-terminal transmembrane domain (TMflex), e.g. lacking aa1148 to aa 1273.

Accordingly, any amino acid sequences being identical or at least 50%,60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 1-26,274-1278, 13521-13587, 22732, 22737-22758, 22929-22964 can be modifiedto remove the endogenous transmembrane domain TM at position aa 1212 toaa 1273 and may therefore be used as “C-terminally truncated” proteinsin the context of the invention (Amino acid positions according toreference SEQ ID NO: 1). Furthermore, any amino acid sequences beingidentical or at least 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one ofSEQ ID NOs: 1-26, 274-1278, 13521-13587, 22732, 22737-22758, 22929-22964can be modified to remove a part the endogenous transmembrane domain(TMflex) at position aa 1148 to aa 1273 and may therefore be used as“C-terminally truncated” proteins in the context of the invention (Aminoacid positions according to reference SEQ ID NO: 1). Suitable spikeproteins lacking the C-terminal transmembrane domain (TM or TMflex) maybe selected from SEQ ID NOs: 31-39, 1614-3623, 13377-13510.

In other embodiments, where the coding sequence of the nucleic acidadditionally encodes heterologous antigen clustering element as definedabove, it is particularly preferred and suitable to generate a fusionprotein comprising an antigen clustering element and an antigenicpeptide or protein derived from SARS-CoV-2 spike protein fragment S1(lacking S2 and/or TM and/or TMflex). Further, it may be suitable to uselinker elements for separating the heterologous antigen-clusteringelement from the antigenic peptide or protein (e.g. a linker accordingto SEQ ID NO: 115, 13148, 13152).

In preferred embodiments, the at least one antigenic peptide or proteincomprising a heterologous antigen clustering element comprises orconsists of at least one of the amino acid sequences being identical orat least 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs:58-75, 85-102, 3624-5633, 7644-9653, 13588-13721, 13856-13989, 22733,22735, 22736 or an immunogenic fragment or immunogenic variant of any ofthese. Further information regarding said amino acid sequences is alsoprovided in Table 1 (see rows 16 and 25 of Column A and B), and under<223> identifier of the ST25 sequence listing of respective sequence SEQID NOs.

Further suitable multimerization elements may be selected from the listof amino acid sequences according to SEQ ID NOs: 1116-1167 ofWO2017/081082, or fragments or variants of these sequences. SEQ ID NOs:1116-1167 of WO2017/081082 are herewith incorporated by reference.

Trimerization Elements

In preferred embodiments, the trimerization element may be selected froma foldon element. In preferred embodiments, the foldon element is afibritin foldon element. Expressing a stable trimeric spike protein,preferably in its prefusion conformation, may increases the magnitudeand breadth of neutralizing activity against SARS-CoV-2.

In particularly preferred embodiments, a fibritin foldon element is usedto promote the antigen trimerization and may therefore promote immuneresponses of the encoded coronavirus antigen, preferably spike protein.Preferably, the foldon element is or is derived from a bacteriophage,preferably from bacteriophage T4, most preferably from fibritin ofbacteriophage T4.

In particularly preferred embodiments, the trimerization element is oris derived from foldon wherein the amino acid sequences of saidtrimerization element is preferably identical or at least 70%, 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to any one of amino acid sequence SEQ ID NO: 114, a fragmentor variant of any of these.

In other embodiments where the coding sequence of the nucleic acidadditionally encodes heterologous trimerization element, it isparticularly preferred and suitable to generate a fusion proteincomprising a trimerization element and an antigenic peptide or proteinderived from SARS-CoV-2. Suitably, said antigenic peptide or protein,preferably the spike protein derived from SARS-CoV-2 that is lacking theC-terminal transmembrane domain (lacking aa 1212 to aa 1273), or islacking a part of the C-terminal transmembrane domain (TMflex), e.g.lacking aa 1148 to aa 1273.

Accordingly, any amino acid sequences being identical or at least 50%,60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 1-26,274-1278, 13521-13587, 22732, 22737-22758, 22947-22964 can be modifiedto lack the endogenous transmembrane element at position aa 1212 to aa1273 and may therefore be used as “C-terminally truncated” proteins inthe context of the invention. Furthermore, any amino acid sequencesbeing identical or at least 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any oneof SEQ ID NOs: 1-26, 274-1278, 13521-13587, 22732, 22737-22758,22947-22964 can be modified to remove a part the endogenoustransmembrane domain (TMflex) at position aa 1148 to aa 1273 and maytherefore be used as “C-terminally truncated” proteins in the context ofthe invention (Amino acid positions according to reference SEQ ID NO:1). Suitable spike proteins lacking the C-terminal transmembrane domain(TM or TMflex) may be selected from SEQ ID NOs: 31-39, 1614-3623,13377-13510.

In other embodiments, where the coding sequence of the nucleic acidadditionally encodes heterologous trimerization element as definedabove, it is particularly preferred and suitable to generate a fusionprotein comprising an trimerization element and an antigenic peptide orprotein derived from SARS-CoV-2 spike protein fragment S1 (lacking S2and/or TM and/or TMflex). Further, it may be suitable to use linkerelements for separating the heterologous antigen-clustering element fromthe antigenic peptide or protein (e.g. a linker according to SEQ ID NO:115, 13148, 13152).

In preferred embodiments, the at least one antigenic peptide or proteincomprising a heterologous trimerization element comprises or consists ofat least one of the amino acid sequences being identical or at least50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 76-84,103-111, 5634-6638, 9654-10658, 13722-13788, 13990-14056, 22734 or animmunogenic fragment or immunogenic variant of any of these. Furtherinformation regarding said amino acid sequences is also provided inTable 1 (see rows 26, 30, and 41 of Column A and B), and under <223>identifier of the ST25 sequence listing of respective sequence SEQ IDNOs.

Further suitable trimerization elements may be selected from the list ofamino acid sequences according to SEQ ID NOs: 1116-1167 ofWO2017/081082, or fragments or variants of these sequences. SEQ ID NOs:1116-1167 of WO2017/081082 are herewith incorporated by reference.

VLP Forming Elements

In preferred embodiments, the VLP forming sequence may be selected andfused to the coronavirus antigen as defined herein. Expressing a stablyclustered spike protein in VLP form may increases the magnitude andbreadth of neutralizing activity against SARS-CoV-2. VLPs structurallymimic infectious viruses and they can induce potent cellular and humoralimmune responses.

Suitable VLP forming sequences may be selected from elements derivedfrom Hepatitis B virus core antigen, HIV-1 Gag protein, or Woodchuckhepatitis core antigen element (WhcAg).

In particularly preferred embodiments, the at least one VLP-formingsequence is a Woodchuck hepatitis core antigen element (WhcAg). TheWhcAg element is used to promote VLP formation and may therefore promoteimmune responses of the encoded coronavirus antigen, preferably spikeprotein.

In particularly preferred embodiments, the VLP forming sequence is or isderived from foldon wherein the amino acid sequences of said VLP formingsequences is preferably identical or at least 70%, 80%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identicalto any one of amino acid sequence SEQ ID NO: 13171, a fragment orvariant of any of these.

In further embodiments where the coding sequence of the nucleic acidadditionally encodes heterologous VLP forming sequence, it isparticularly preferred and suitable to generate a fusion proteincomprising a VLP forming sequence and an antigenic peptide or proteinderived from SARS-CoV-2. Suitably, said antigenic peptide or protein,preferably the spike protein derived from SARS-CoV-2 that is lacking theC-terminal transmembrane domain (lacking aa 1212 to aa 1273), or islacking a part of the C-terminal transmembrane domain (TMflex), e.g.lacking aa 1148 to aa 1273.

Accordingly, any amino acid sequences being identical or at least 50%,60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 1-26,274-1278, 13521-13587, 22732, 22737-22758, 22929-22964 can be modifiedto lack the endogenous transmembrane element at position aa 1212 to aa1273 and may therefore be used as “C-terminally truncated” proteins inthe context of the invention. Furthermore, any amino acid sequencesbeing identical or at least 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any oneof SEQ ID NOs: 1-26, 274-1278, 13521-13587, 22732, 22737-22758,22929-22964 can be modified to remove a part the endogenoustransmembrane domain (TMflex) at position aa 1148 to aa 1273 and maytherefore be used as “C-terminally truncated” proteins in the context ofthe invention (Amino acid positions according to reference SEQ ID NO:1). Suitable spike proteins lacking the C-terminal transmembrane domain(TM or TMflex) may be selected from SEQ ID NOs: 31-39, 1614-3623,13377-13510.

In other embodiments, where the coding sequence of the nucleic acidadditionally encodes heterologous VLP-forming sequence as defined above,it is particularly preferred and suitable to generate a fusion proteincomprising a VLP-forming sequence and an antigenic peptide or proteinderived from SARS-CoV-2 spike protein fragment S1 (lacking S2 and/or TMand/or TMflex). Further, it may be suitable to use linker elements forseparating the heterologous antigen-clustering element from theantigenic peptide or protein (e.g. a linker according to SEQ ID NO: 115,13148, 13152).

In preferred embodiments, the at least one antigenic peptide or proteincomprising a heterologous VLP-forming sequence comprises or consists ofat least one of the amino acid sequences being identical or at least50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs:6639-7643, 10659-11663, 13789-13855, 14057-14123 or an immunogenicfragment or immunogenic variant of any of these. Further informationregarding said amino acid sequences is also provided in Table 1 (seerows 31 and 35 of Column A and B), and under <223> identifier of theST25 sequence listing of respective sequence SEQ ID NOs.

Further suitable VLP forming sequences in that context may be selectedfrom the list of amino acid sequences according to SEQ ID NOs: 1168-1227of the patent application WO2017/081082, or fragments or variants ofthese sequences. SEQ ID NOs: 1168-1227 of WO2017/081082 are herewithincorporated by reference.

Heterologous Secretory Signal Peptides

In some embodiments, the antigenic peptide or protein comprises aheterologous signal peptide. A heterologous signal peptide may be usedto improve the secretion of the encoded coronavirus antigen.

Suitable secretory signal peptides may be selected from the list ofamino acid sequences according to SEQ ID NOs: 1-1115 and SEQ ID NO: 1728of published PCT patent application WO2017/081082, or fragments orvariants of these sequences. 1-1115 and SEQ ID NO: 1728 of WO2017/081082are herewith incorporated by reference. In embodiments where the codingsequence of the nucleic acid additionally encodes heterologous secretorysignal peptide, it is particularly preferred and suitable to generate afusion protein comprising a heterologous secretory signal peptide and anantigenic peptide or protein derived from SARS-CoV-2. Suitably, saidantigenic peptide or protein, preferably the spike protein derived fromSARS-CoV-2 is lacking the N-terminal endogenous secretory signal peptide(lacking aa 1 to aa 15). Accordingly, any amino acid sequences beingidentical or at least 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one ofSEQ ID NOs: 1-26, 274-1278, 13521-13587, 22732, 22737-22758, 22929-22964can be modified to lack the endogenous secretory signal peptide atposition aa 1 to aa 15 and may therefore be used as “N-terminallytruncated” proteins in the context of the invention.

In the following List 1, suitable SARS-CoV-2 coronavirus antigenicpeptides and proteins as defined above are further specified in detail(e.g. nomenclature, protein elements, etc.).

List 1: Exemplary Suitable Protein Designs of the Invention:

-   -   Full length spike protein (S) comprising aa 1 to aa 1273;        -   see for example SEQ ID NO: 1, 274.    -   Stabilized S protein comprising aa1-aa1273 and K986P, V987P        substitutions (S_stab_PP);        -   see for example SEQ ID NO: 10, 341.    -   Stabilized S protein comprising aa1-aa1273 and K986P, V987P        substitutions (S_stab_PP);        -   see for example SEQ ID NO: 22961.    -   Stabilized S protein comprising aa1-aa1273 and K986P, V987P        substitutions (S_stab_PP);        -   see for example SEQ ID NO: 22960.    -   Stabilized S protein comprising aa1-aa1273 and K986P, V987P,        F817P, A892P, A899P, A942P proline substitutions; S_stab_PP_hex        -   see for example SEQ ID NO: 22732.    -   Stabilized S protein comprising aa1-aa1273 and K986P, V987P        substitutions and a cavity filling mutation (T887W, A1020W);        S_stab_PP_cav        -   see for example SEQ ID NO: 408.    -   Stabilized S protein comprising aa1-aa1273 and K986P, V987P        substitutions and a cavity filling mutation (P1069F);        S_stab_PP_cav        -   see for example SEQ ID NO: 475.    -   Stabilized S protein comprising aa1-aa1273 and K986P, V987P        substitutions and a cavity filling mutation (H1048Q, H1064N,        H1083N, H1101N); S_stab_PP_prot        -   see for example SEQ ID NO: 542.    -   Stabilized S protein comprising aa1-aa1273 and an artificial        disulfide bond (S_stab_disul) 17120, T1077C;        -   see for example SEQ ID NO: 19, 609.    -   S without transmembrane domain comprising aa1-aa1211 (S_woTM);        -   see for example SEQ ID NO: 31, 1614.    -   S without transmembrane domain flex comprising aa1-aa1147        (S_woTMflex);        -   see for example SEQ ID NO: 2619.    -   S_woTM comprising K986P, V987P substitutions (S_stab_PP_woTM)        -   see for example SEQ ID NO: 40, 1681.    -   S_woTMflex comprising K986P, V987P substitutions        (S_stab_PP_woTMflex)        -   see for example SEQ ID NO: 2686.    -   Spike protein fragment S1 comprising aa 1 to aa 681 (S1);        -   see for example SEQ ID NO: 27, 1279.    -   S_woTM comprising a lumazine synthase;        -   see for example SEQ ID NO: 58, 3624.    -   S_woTMflex comprising a lumazine synthase;        -   see for example SEQ ID NO: 7644.    -   S_stab_PP_woTM comprising a lumazine synthase;        -   see for example SEQ ID NO: 85, 3691.    -   S_stab_PP_woTMflex comprising a lumazine synthase;        -   see for example SEQ ID NO: 7711.    -   S_woTM comprising a ferritin element;        -   see for example SEQ ID NO: 67, 4629.    -   S_woTMflex comprising a ferritin element;        -   see for example SEQ ID NO: 8649.    -   S_stab_PP_woTM comprising a ferritin element;        -   see for example SEQ ID NO: 94, 4696.    -   S_stab_PP_woTMflex comprising a ferritin element;        -   see for example SEQ ID NO: 8716.    -   S_woTM comprising a foldon element;        -   see for example SEQ ID NO: 76, 5634.    -   S_woTMflex comprising a foldon element;        -   see for example SEQ ID NO: 9654.    -   S_stab_PP_woTM comprising a foldon element;        -   see for example SEQ ID NO: 103, 5701.    -   S_stab_PP_woTMflex comprising a foldon element;        -   see for example SEQ ID NO: 9721.    -   S_woTM comprising a VLP-sequence (WhcAg);        -   see for example SEQ ID NO: 6639.    -   S_woTMflex comprising a VLP-sequence (WhcAg);        -   see for example SEQ ID NO 10659.    -   S_stab_PP_woTM comprising a VLP-sequence (WhcAg);        -   see for example SEQ ID NO: 6706.    -   S_stab_PP_woTMflex comprising a VLP-sequence (WhcAg);        -   see for example SEQ ID NO: 10726.    -   truncRBD comprising a foldon element:        -   see for example SEQ ID NO: 22734.    -   truncRBD comprising a lumazine synthase (C-terminal)        -   see for example SEQ ID NO: 22735.    -   truncRBD comprising a lumazine synthase (N-terminal)        -   see for example SEQ ID NO: 22736    -   truncRBD comprising a ferritin element:        -   see for example SEQ ID NO: 22733.

Amino acid positions provided in List 1 are according to reference SEQID NO: 1.

In particularly preferred embodiments of the first aspect, the at leastone antigenic peptide or protein comprises or consists of at least oneof the amino acid sequences being identical or at least 70%, 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to any one of SEQ ID NOs: 10, 21, 22, 25, 27, 274, 341, 408,475, 542, 743, 810, 1011, 1145, 1212, 1279, 8716, 10726, 22732-22758,22929-22942, 22947-22964 or an immunogenic fragment or immunogenicvariant of any of these.

Preferred antigenic peptide or proteins derived from an SARS-CoV-2coronavirus as defined above are provided in Table 1 (rows 1 to 41).Therein, each row 1 to 41 corresponds to a suitable SARS-CoV-2coronavirus constructs. Column A of Table 1 provides a short descriptionof suitable antigen constructs. Column B of Table 1 provides protein(amino acid) SEQ ID NOs of respective antigen constructs. Column C ofTable 1 provides SEQ ID NO of the corresponding wild type nucleic acidcoding sequences. Column D of Table 1 provides SEQ ID NO of thecorresponding G/C optimized nucleic acid coding sequences (opt1, gc).Column E of Table 1 provides SEQ ID NO of the corresponding human codonusage adapted nucleic acid coding sequences (opt 3, human). Column F ofTable 1 provides SEQ ID NO of the corresponding G/C content modifiednucleic acid coding sequences (opt10, gc mod) (for a detaileddescription of “coding sequences”, see paragraph “suitable codingsequences”).

Notably, the description of the invention explicitly includes theinformation provided under <223> identifier of the ST25 sequence listingof the present application. Preferred nucleic acid constructs comprisingcoding sequences of Table 1, e.g. mRNA sequences comprising the codingsequences of Table 1 are provided in Table 3a and Table 3b.

TABLE 1 Preferred coronavirus constructs (amino acid sequences andnucleic acid coding sequences): row A B C D E F 1 Full-length spikeprotein; S 1-9, 274-340, 116-131, 136, 11731- 11967- 12034; 22737,22739,11664- 11797,22764, 12033 23041-23076 22741,22743, 11730 22766, 22768,22745, 22747, 22770, 22772, 22749, 22751, 22774, 22776, 22753, 22755,22778, 22780, 22757; 22929- 22782, 22784; 22946 22969-23040 2 Stabilizedspike protein; 10-18, 341- 137, 11798, 142 146, 12035; S_stab_PP 407,22738, 22765, 22767, 23149-23184 22740, 22742, 22769, 22771, 22744,22746, 22773, 22775, 22748, 22750, 22777, 22779, 22752, 22754, 22781,22783, 22756, 22758; 22785; 23077- 22947-22964 23148 3 Stabilized spikeprotein; 408-541 11799, 11800 12036, 12037 S_stab_PP_cav 4 Stabilizedspike protein; 542-608 11801 12038 S_stab_PP_prot 5 Stabilized spikeprotein; 19-26, 609- 11802-11811, 12039-12048, S_stab_disul 1278, 13521-14124 14133 13587 6 Spike protein fragment S1 27,1279-1345 132 138,11812 143 147, 12049 7 Spike protein fragment S2 30, 1346-1412 135 141,11813 12050 8 Signal peptide of spike protein; 28,1413-1479 133 139,11814 144 12051 SP 9 S1 without signal peptide; 29,1480-1546 134 140,11815 145 12052 S1_woSP 10 Transmembrane domain of 49-57, 1547- 11816,13511 12053, 13516 spike protein; TM/TMflex 1613, 13176- 13242 11 Swithout transmembrane 31-39, 1614- 11817, 11832 12054, 12069 domain;S_woTM/woTMflex 1680, 2619- 2685 12 Stabilized S without 40-48, 1681-11818, 11833 12055, 12070 transmembrane domain; 1747, 2686-S_stab_PP_woTM/woTMflex 2752 13 Stabilized S without 1748-1881,11819,11820, 12056, transmembrane domain; 2753-2886 11834, 11835 12057,S_stab_PP_cav_woTM/ 12071, 12072 woTMflex 14 Stabilized S without1882-1948, 11821, 11836 12058, 12073 transmembrane domain; 2887-2953S_stab_PP_prot_woTM/ woTMflex 15 Stabilized S without 1949-2618,11822-11831, 12059-12068, transmembrane domain; 2954-3623, 11837-11846,12074-12083, S_stab_disul_woTM/woTMflex 13377-13510 13514, 13515 13519,13520 16 S_woTM/woTMflex comprising 58-66, 3624- 11847, 11907 12084,12144 a lumazine synthase 3690, 7644- 7710 17 S_stab_PP_woTM/woTMflex85-93, 3691- 11848, 11908 12085, 12145 comprising a lumazine synthase3757, 7711- 7777 18 S_stab_PP_cav_woTM/ 3758-3891, 11849, 11850, 12086,woTMflex comprising a 7778-7911 11909, 11910 12087, lumazine synthase12146, 12147 19 S_stab_PP_prot_woTM/ 3892-3958, 11851, 11911 12088,12148 woTMflex comprising a 7912-7978 lumazine synthase 20S_stab_disul_woTM/woTMflex 3959-4628, 11852-11861, 12089-12098,comprising a lumazine synthase 7979-8648, 11912-11921, 12149-12158,14125, 14129 14134, 14138 13588-13654, 13856-13922 21 S_woTM/woTMflexcomprising 67-75, 4629- 11862, 11922 12099, 12159 a ferritin 4695, 8649-8715 22 S_stab_PP_woTM/woTMflex 94-102, 4696- 11863, 11923 12100, 12160comprising a ferritin 4762, 8716- 8782 23 S_stab_PP_cav_woTM/ 4763-4896,11864,11865, 12101, woTMflex comprising a ferritin 8783-8916 11924,11925 12102, 12161, 12162 24 S_stab_PP_prot_woTM/ 4897-4963, 11866,11926 12103, 12163 woTMflex comprising a ferritin 8917-8983 25S_stab_disul_woTM/woTMflex 4964-5633, 11867-11876, 12104-12113,comprising a ferritin 8984-9653, 11927-11936, 12164-12173, 13655-13721,14126, 14130 14135, 14139 13923-13989 26 S_woTM/woTMflex comprising76-84, 5634- 11877, 11937 12114, 12174 a foldon 5700, 9654- 9720 27S_stab_ PP_woTM/woTMflex 103-111, 11878, 11938 12115, 12175 comprising afoldon 5701-5767, 9721-9787 28 S_stab_PP_cav_woTM/ 5768-5901, 11879,11880, 12116, woTMflex comprising a foldon 9788-9921 11939, 11940 12117,12176, 12177 29 S_stab_PP_prot_woTM/ 5902-5968, 11881, 11941 12118,12178 woTMflex comprising a foldon 9922-9988 30S_stab_disul_woTM/woTMflex 5969-6638, 11882-11891, 12119-12128,comprising a foldon 9989-10658, 11942-11951, 12179-12188, 13722-13788,14127, 14131 14136, 14140 13990-14056 31 S_woTM/woTMflex comprising6639-6705, 11892, 11952 12129, 12189 a WhcAg (VLP) 10659-10725 32S_stab_PP_woTM/woTMflex 6706-6772, 11893, 11953 12130, 12190 comprisinga WhcAg (VLP) 10726-10792 33 S_stab_PP_cav_woTM/ 6773-6906, 11894,11895,12131, woTMflex comprising a WhcAg 10793-10926 11954, 11955 12132, (VLP)12191, 12192 34 S_stab_PP_prot_woTM/ 6907-6973, 11896, 11956 12133,12193 woTMflex comprising a WhcAg 10927-10993 (VLP) 35S_stab_disul_woTM/woTMflex 6974-7643, 11897-11906, 12134-12143,comprising a WhcAg (VLP) 10994-11663, 11957-11966, 12194-12203,13789-13855, 14128, 14132 14137, 14141 14057-14123 36 Receptor bindingdomain; RBD 13243-13309, 13512, 22914, 13517, 22917, 22923 22918, 22919,22915, 22924, 22925 22916, 22920-22922, 22926-22928 37 Criticalneutralisation domain; 13310-13376 13513 13518 CND 38 Stabilized spikeprotein; 22732 22759 S_stab_PP_hex 39 RBD comprising a lumazyne 22735,22736 22762, 22763, synthase 22967, 22968 40 RBD comprising a ferritin22733 22760, 22965 41 RBD comprising a foldon 22734, 26938, 22761,22966, 26939 26940, 26941

Suitable Coding Sequences:

According to preferred embodiments, the nucleic acid of the inventioncomprises at least one coding sequence encoding at least one antigenicpeptide or protein derived from SARS-CoV-2 (nCoV-2019) coronavirus,preferably as defined above, or fragments and variants thereof. In thatcontext, any coding sequence encoding at least one antigenic protein asdefined herein, or fragments and variants thereof may be understood assuitable coding sequence and may therefore be comprised in the nucleicacid of the invention.

In preferred embodiments, the nucleic acid of the first aspect maycomprise or consist of at least one coding sequence encoding at leastone antigenic peptide or protein from SARS-CoV-2 coronavirus as definedherein, preferably encoding any one of SEQ ID NOs: 1-111, 274-11663,13176-13510, 13521-14123, 22732-22758, 22917, 22923, 22929-22964, 26938,26939 or fragments of variants thereof. It has to be understood that, onnucleic acid level, any sequence (DNA or RNA sequence) which encodes anamino acid sequences being identical or at least 70%, 80%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to any one of SEQ ID NOs: 1-111, 274-11663, 13176-13510,13521-14123, 22732-22758, 22917, 22923, 22929-22964, 26938, 26939 orfragments or variants thereof, may be selected and may accordingly beunderstood as suitable coding sequence of the invention. Furtherinformation regarding said amino acid sequences is also provided inTable 1 (see rows 1 to 41 of Column A and B), Table 3a and 3b, and under<223> identifier of the ST25 sequence listing of respective sequence SEQID NOs.

In preferred embodiments, the nucleic acid of the first aspect comprisesa coding sequence that comprises at least one of the nucleic acidsequences being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to thesequences according to SEQ ID NOs: 116-132, 134-138, 140-143, 145-175,11664-11813, 11815, 11817-12050, 12052, 12054-13147, 13514, 13515,13519, 13520, 14124-14177, 22759, 22764-22786, 22791-22813, 22818-22839,22969-23184, 23189-23404, 23409-23624, 23629-23844, 23849-24064,24069-24284, 24289-24504, 24509-24724, 24729-24944, 24949-25164,25169-25384, 25389-25604, 25609-25824, 25829-26044, 26049-26264,26269-26484, 26489-26704, 26709-26937 or a fragment or a fragment orvariant of any of these sequences. Further information regarding saidnucleic acid sequences is also provided in Table 1 (see rows 1 to 7, 9,11-41 of Column C-F), Table 3a and 3b, and under <223> identifier of theST25 sequence listing of respective sequence SEQ ID NOs.

Alternatively, the nucleic acid of the first aspect comprises a codingsequence that comprises at least one of the nucleic acid sequences beingidentical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequencesaccording SEQ ID NOs: 116-132, 134-138, 140-143, 145-175, 11664-11813,11815, 11817-12050, 12052, 12054-13147, 13514, 13515, 13519, 13520,14124-14177, 22759, 22764-22786, 22791-22813, 22818-22839, 22969-23184,23189-23404, 23409-23624, 23629-23844, 23849-24064, 24069-24284,24289-24504, 24509-24724, 24729-24944, 24949-25164, 25169-25384,25389-25604, 25609-25824, 25829-26044, 26049-26264, 26269-26484,26489-26704, 26709-26937 wherein all Uracils (U) in the respectivesequences are substituted by Thymidines (T), or a fragment or a fragmentor variant of any of these sequences. Further information regarding saidnucleic acid sequences is also provided in Table 1 (see rows 1 to 7, 9,11-41 of Column C-F), Table 3a and 3b, and under <223> identifier of theST25 sequence listing of respective sequence SEQ ID NOs.

In preferred embodiments, the nucleic acid of the first aspect comprisesa coding sequence that comprises at least one of the nucleic acidsequences being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to thesequences according to SEQ ID NOs: 116-132, 134-138, 140-143, 145-175,11664-11813, 11815, 11817-12050, 12052, 12054-12203, 13514, 13515,13519, 13520, 14124-14141, 22759, 22764-22785, 22969-23184 or a fragmentor a fragment or variant of any of these sequences. Further informationregarding said nucleic acid sequences is also provided in Table 1 (seerows 1 to 7, 9, 11-41 of Column C-F), Table 3a and 3b, and under <223>identifier of the ST25 sequence listing of respective sequence SEQ IDNOs.

Alternatively, the nucleic acid of the first aspect comprises a codingsequence that comprises at least one of the nucleic acid sequences beingidentical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequencesaccording SEQ ID NOs: 116-132, 134-138, 140-143, 145-175, 11664-11813,11815, 11817-12050, 12052, 12054-12203, 13514, 13515, 13519, 13520,14124-14141, 22759, 22764-22785, 22969-23184 wherein all Uracils (U) inthe respective sequences are substituted by Thymidines (T), or afragment or a fragment or variant of any of these sequences. Furtherinformation regarding said nucleic acid sequences is also provided inTable 1 (see rows 1 to 7, 9, 11-41 of Column C-F), Table 3a and 3b, andunder <223> identifier of the ST25 sequence listing of respectivesequence SEQ ID NOs.

In preferred embodiments, the nucleic acid of the first aspect is anartificial nucleic acid, e.g. an artificial DNA or an artificial RNA.

The term “artificial nucleic acid” as used herein is intended to referto a nucleic acid that does not occur naturally. In other words, anartificial nucleic acid may be understood as a non-natural nucleic acidmolecule. Such nucleic acid molecules may be non-natural due to itsindividual sequence (e.g. G/C content modified coding sequence, UTRs)and/or due to other modifications, e.g. structural modifications ofnucleotides. Typically, artificial nucleic acid may be designed and/orgenerated by genetic engineering to correspond to a desired artificialsequence of nucleotides. In this context, an artificial nucleic acid isa sequence that may not occur naturally, i.e. a sequence that differsfrom the wild type sequence/the naturally occurring sequence by at leastone nucleotide. The term “artificial nucleic acid” is not restricted tomean “one single molecule” but is understood to comprise an ensemble ofessentially identical nucleic acid molecules. Accordingly, it may relateto a plurality of essentially identical nucleic acid molecules. The term“artificial nucleic acid” as used herein may relate to an artificial DNAor, preferably, to an artificial RNA.

In preferred embodiments, the nucleic acid, preferably the DNA or RNA ofthe first aspect is a modified and/or stabilized nucleic acid,preferably a modified and/or stabilized artificial nucleic acid.

According to preferred embodiments, the nucleic acid of the presentinvention may thus be provided as a “stabilized artificial nucleic acid”or “stabilized coding nucleic acid” that is to say a nucleic acidshowing improved resistance to in vivo degradation and/or a nucleic acidshowing improved stability in vivo, and/or a nucleic acid showingimproved translatability in vivo. In the following, specific suitablemodifications/adaptations in this context are described which aresuitably to “stabilize” the nucleic acid. Preferably, the nucleic acidof the present invention may be provided as a “stabilized RNA”,“stabilized coding RNA”, “stabilized DNA” or “stabilized coding DNA”.

Such stabilization may be effected by providing a “dried nucleic acid”(e.g. a dried DNA or RNA) and/or a “purified nucleic acid” (e.g. apurified DNA or RNA) as specified herein. Alternatively or in additionto that, such stabilization can be effected, for example, by a modifiedphosphate backbone of the nucleic acid of the present invention. Abackbone modification in connection with the present invention is amodification in which phosphates of the backbone of the nucleotidescontained in the nucleic acid are chemically modified. Nucleotides thatmay be preferably used in this connection contain e.g. aphosphorothioate-modified phosphate backbone, preferably at least one ofthe phosphate oxygens contained in the phosphate backbone being replacedby a sulfur atom. Stabilized nucleic acids, preferably stabilized RNAsmay further include, for example: non-ionic phosphate analogues, suchas, for example, alkyl and aryl phosphonates, in which the chargedphosphonate oxygen is replaced by an alkyl or aryl group, orphosphodiesters and alkylphosphotriesters, in which the charged oxygenresidue is present in alkylated form. Such backbone modificationstypically include, without implying any limitation, modifications fromthe group consisting of methylphosphonates, phosphoramidates andphosphorothioates (e.g. cytidine-5′-O-(1-thiophosphate)).

In the following, suitable modifications are described that are capableof “stabilizing” the nucleic acid of the invention.

In preferred embodiments, the nucleic acid, e.g. the RNA or DNA,comprises at least one codon modified coding sequence.

In preferred embodiments, the at least one coding sequence of thenucleic acid is a codon modified coding sequence, wherein the amino acidsequence encoded by the at least one codon modified coding sequence ispreferably not being modified compared to the amino acid sequenceencoded by the corresponding wild type or reference coding sequence.

The term “codon modified coding sequence” relates to coding sequencesthat differ in at least one codon (triplets of nucleotides coding forone amino acid) compared to the corresponding wild type or referencecoding sequence. Suitably, a codon modified coding sequence in thecontext of the invention may show improved resistance to in vivodegradation and/or improved stability in vivo, and/or improvedtranslatability in vivo. Codon modifications in the broadest sense makeuse of the degeneracy of the genetic code wherein multiple codons mayencode the same amino acid and may be used interchangeably (cf. Table 2)to optimize/modify the coding sequence for in vivo applications asoutlined above.

The term “reference coding sequence” relates to the coding sequence,which was the origin sequence to be modified and/or optimized.

In preferred embodiments, the at least one coding sequence of thenucleic acid is a codon modified coding sequence, wherein the codonmodified coding sequence is selected from C maximized coding sequence,CAI maximized coding sequence, human codon usage adapted codingsequence, G/C content modified coding sequence, and G/C optimized codingsequence, or any combination thereof.

When transfected into mammalian host cells, the nucleic acid comprisinga codon modified coding sequence has a stability of between 12-18 hours,or greater than 18 hours, e.g., 24, 36, 48, 60, 72, or greater than 72hours and are capable of being expressed by the mammalian host cell(e.g. a muscle cell).

When transfected into mammalian host cells, the nucleic acid comprisinga codon modified coding sequence is translated into protein, wherein theamount of protein is at least comparable to, or preferably at least 10%more than, or at least 20% more than, or at least 30% more than, or atleast 40% more than, or at least 50% more than, or at least 100% morethan, or at least 200% or more than the amount of protein obtained by anaturally occurring or wild type or reference coding sequencetransfected into mammalian host cells.

In preferred embodiments, the nucleic acid of the invention may bemodified, wherein the C content of the at least one coding sequence maybe increased, preferably maximized, compared to the C content of thecorresponding wild type or reference coding sequence (herein referred toas “C maximized coding sequence”). The amino acid sequence encoded bythe C maximized coding sequence of the nucleic acid is preferably notmodified compared to the amino acid sequence encoded by the respectivewild type or reference coding sequence. The generation of a C maximizednucleic acid sequences may suitably be carried out using a modificationmethod according to WO2015/062738. In this context, the disclosure ofWO2015/062738 is included herewith by reference.

In preferred embodiments, the nucleic acid may be modified, wherein theG/C content of the at least one coding sequence may be optimizedcompared to the G/C content of the corresponding wild type or referencecoding sequence (herein referred to as “G/C content optimized codingsequence”). “Optimized” in that context refers to a coding sequencewherein the G/C content is preferably increased to the essentiallyhighest possible G/C content. The amino acid sequence encoded by the G/Ccontent optimized coding sequence of the nucleic acid is preferably notmodified as compared to the amino acid sequence encoded by therespective wild type or reference coding sequence. The generation of aG/C content optimized nucleic acid sequence (RNA or DNA) may be carriedout using a method according to WO2002/098443. In this context, thedisclosure of WO2002/098443 is included in its full scope in the presentinvention. Throughout the description, including the <223> identifier ofthe sequence listing, G/C optimized coding sequences are indicated bythe abbreviations “opt1” or “gc”.

In preferred embodiments, the nucleic acid may be modified, wherein thecodons in the at least one coding sequence may be adapted to human codonusage (herein referred to as “human codon usage adapted codingsequence”). Codons encoding the same amino acid occur at differentfrequencies in humans. Accordingly, the coding sequence of the nucleicacid is preferably modified such that the frequency of the codonsencoding the same amino acid corresponds to the naturally occurringfrequency of that codon according to the human codon usage. For example,in the case of the amino acid Ala, the wild type or reference codingsequence is preferably adapted in a way that the codon “GCC” is usedwith a frequency of 0.40, the codon “GCT” is used with a frequency of0.28, the codon “GCA” is used with a frequency of 0.22 and the codon“GCG” is used with a frequency of 0.10 etc. (see Table 2). Accordingly,such a procedure (as exemplified for Ala) is applied for each amino acidencoded by the coding sequence of the nucleic acid to obtain sequencesadapted to human codon usage. Throughout the description, including the<223> identifier of the sequence listing, human codon usage adaptedcoding sequences are indicated by the abbreviation “opt3” or “human”.

TABLE 2 Human codon usage table with frequencies indicated for eachamino acid Amino acid codon frequency Amino acid codon frequency Ala GCG0.10 Pro CCG 0.11 Ala GCA 0.22 Pro CCA 0.27 Ala GCT 0.28 Pro CCT 0.29Ala GCC* 0.40 Pro CCC* 0.33 Cys TGT 0.42 Gln CAG* 0.73 Cys TGC* 0.58 GlnCAA 0.27 Asp GAT 0.44 Arg AGG 0.22 Asp GAC* 0.56 Arg AGA* 0.21 Glu GAG*0.59 Arg CGG 0.19 Glu GAA 0.41 Arg CGA 0.10 Phe TTT 0.43 Arg CGT 0.09Phe TTC* 0.57 Arg CGC 0.19 Gly GGG 0.23 Ser AGT 0.14 Gly GGA 0.26 SerAGC* 0.25 Gly GGT 0.18 Ser TCG 0.06 Gly GGC* 0.33 Ser TCA 0.15 His CAT0.41 Ser TCT 0.18 His CAC* 0.59 Ser TCC 0.23 Ile ATA 0.14 Thr ACG 0.12Ile ATT 0.35 Thr ACA 0.27 Ile ATC* 0.52 Thr ACT 0.23 Lys AAG* 0.60 ThrACC* 0.38 Lys AAA 0.40 Val GTG* 0.48 Leu TTG 0.12 Val GTA 0.10 Leu TTA0.06 Val GTT 0.17 Leu CTG* 0.43 Val GTC 0.25 Leu CTA 0.07 Trp TGG* 1 LeuCTT 0.12 Tyr TAT 0.42 Leu CTC 0.20 Tyr TAC* 0.58 Met ATG* 1 Stop TGA*0.61 Asn AAT 0.44 Stop TAG 0.17 Asn AAC* 0.56 Stop TAA 0.22 *mostfrequent human codon

In embodiments, the nucleic acid of the invention may be modified,wherein the G/C content of the at least one coding sequence may bemodified compared to the G/C content of the corresponding wild type orreference coding sequence (herein referred to as “G/C content modifiedcoding sequence”). In this context, the terms “G/C optimization” or “G/Ccontent modification” relate to a nucleic acid that comprises amodified, preferably an increased number of guanosine and/or cytosinenucleotides as compared to the corresponding wild type or referencecoding sequence. Such an increased number may be generated bysubstitution of codons containing adenosine or thymidine nucleotides bycodons containing guanosine or cytosine nucleotides. Advantageously,nucleic acid sequences having an increased G/C content are more stableor show a better expression than sequences having an increased A/U.

The amino acid sequence encoded by the G/C content modified codingsequence of the nucleic acid is preferably not modified as compared tothe amino acid sequence encoded by the respective wild type or referencesequence. Preferably, the G/C content of the coding sequence of thenucleic acid is increased by at least 10%, 20%, 30%, preferably by atleast 40% compared to the G/C content of the coding sequence of thecorresponding wild type or reference nucleic acid sequence (hereinreferred to “opt 10” or “gc mod”)

In embodiments, the nucleic acid may be modified, wherein the codonadaptation index (CAI) may be increased or preferably maximised in theat least one coding sequence (herein referred to as “CAI maximizedcoding sequence”). It is preferred that all codons of the wild type orreference nucleic acid sequence that are relatively rare in e.g. a humanare exchanged for a respective codon that is frequent in the e.g. ahuman, wherein the frequent codon encodes the same amino acid as therelatively rare codon. Suitably, the most frequent codons are used foreach amino acid of the encoded protein (see Table 2, most frequent humancodons are marked with asterisks). Suitably, the nucleic acid comprisesat least one coding sequence, wherein the codon adaptation index (CAI)of the at least one coding sequence is at least 0.5, at least 0.8, atleast 0.9 or at least 0.95. Most preferably, the codon adaptation index(CAI) of the at least one coding sequence is 1 (CAI=1). For example, inthe case of the amino acid Ala, the wild type or reference codingsequence may be adapted in a way that the most frequent human codon“GCC” is always used for said amino acid. Accordingly, such a procedure(as exemplified for Ala) may be applied for each amino acid encoded bythe coding sequence of the nucleic acid to obtain CAI maximized codingsequences.

In particularly preferred embodiments, the at least one coding sequenceof the nucleic acid is a codon modified coding sequence, wherein thecodon modified coding sequence is selected a G/C optimized codingsequence, a human codon usage adapted coding sequence, or a G/C modifiedcoding sequence

In preferred embodiments, the nucleic acid of the first aspect comprisesat least one coding sequence comprising or consisting a codon modifiednucleic acid sequence which is identical or at least 70%, 80%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to a codon modified nucleic acid sequence selected from thegroup consisting of SEQ ID NOs: 136-138, 140-143, 145-175, 11731-11813,11815, 11817-12050, 12052, 12054-13147, 14142-14177, 22759, 22764-22786,22791-22813, 22818-22839, 22969-23184, 23189-23404, 23409-23624,23629-23844, 23849-24064, 24069-24284, 24289-24504, 24509-24724,24729-24944, 24949-25164, 25169-25384, 25389-25604, 25609-25824,25829-26044, 26049-26264, 26269-26484, 26489-26704, 26709-26937 or afragment or variant of any of these sequences. Additional informationregarding each of these suitable nucleic acid sequences encoding mayalso be derived from the sequence listing, in particular from thedetails provided therein under identifier <223>. Suitable codingsequences of the first aspect are provided in Table 1. Furtherinformation regarding said nucleic acid sequences is also provided inTable 1 (see rows 1 to 7, 9, 11-41 of Column D-F), Table 3a and 3b, andunder <223> identifier of the ST25 sequence listing of respectivesequence SEQ ID NOs.

Alternatively, the nucleic acid of the first aspect comprises at leastone coding sequence comprising or consisting a codon modified nucleicacid sequence which is identical or at least 70%, 80%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identicalto the sequences according to SEQ ID NOs: 136-138, 140-143, 145-175,11731-11813, 11815, 11817-12050, 12052, 12054-13147, 14142-14177, 22759,22764-22786, 22791-22813, 22818-22839, 22969-23184, 23189-23404,23409-23624, 23629-23844, 23849-24064, 24069-24284, 24289-24504,24509-24724, 24729-24944, 24949-25164, 25169-25384, 25389-25604,25609-25824, 25829-26044, 26049-26264, 26269-26484, 26489-26704,26709-26937 wherein all Uracils (U) in the respective sequences aresubstituted by Thymidines (T), or a fragment or a fragment or variant ofany of these sequences. Further information regarding said nucleic acidsequences is also provided in Table 1 (see rows 1 to 7, 9, 11-41 ofColumn D-F), Table 3a and 3b, and under <223> identifier of the ST25sequence listing of respective sequence SEQ ID NOs.

In particularly preferred embodiments, the nucleic acid of the firstaspect comprises at least one coding sequence comprising or consisting aG/C optimized coding sequence which is identical or at least 70%, 80%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identical to a codon modified nucleic acid sequence selected fromthe group consisting of SEQ ID NOs: 136-138, 140, 141, 148, 149, 152,155, 156, 159, 162, 163, 166, 169, 170, 173, 11731-11813, 11815,11817-11966, 12271-12472, 12743-12944, 13514, 13515, 14124-14132,14142-14150, 14160-14168, 22759, 22764-22786, 22791-22813, 22818-22839,22969-23040, 23077-23148, 23189-23260, 23297-23368, 23409-23480,23517-23588, 23629-23700, 23737-23808, 23849-23920, 23957-24028,24069-24140, 24177-24248, 24289-24360, 24397-24468, 24509-24580,24617-24688, 24729-24800, 24837-24908, 24949-25020, 25057-25128,25169-25240, 25277-25348, 25389-25460, 25497-25568, 25609-25680,25717-25788, 25829-25900, 25937-26008, 26049-26120, 26157-26228,26269-26340, 26377-26448, 26489-26560, 26597-26668, 26709-26780,26817-26888, 26925-26937 or a fragment or variant of any of thesesequences. Additional information regarding each of these suitablenucleic acid sequences encoding may also be derived from the sequencelisting, in particular from the details provided therein underidentifier <223>. Suitable coding sequences of the first aspect areprovided in Table 1. Further information regarding said nucleic acidsequences is also provided in Table 1 (see rows 1 to 7, 9, 11-41 ofColumn D), Table 3a and 3b, and under <223> identifier of the ST25sequence listing of respective sequence SEQ ID NOs.

In particularly preferred embodiments, the nucleic acid of the firstaspect comprises at least one coding sequence comprising or consisting ahuman codon usage adapted coding sequence which is identical or at least70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% identical to a codon modified nucleic acid sequenceselected from the group consisting of SEQ ID NOs: 142, 143, 145, 150,153, 157, 160, 164, 167, 171, 174, 11967-12033, 12473-12539, 12945-13011or a fragment or variant of any of these sequences. Additionalinformation regarding each of these suitable nucleic acid sequencesencoding may also be derived from the sequence listing, in particularfrom the details provided therein under identifier <223>. Suitablecoding sequences of the first aspect are provided in Table 1. Furtherinformation regarding said nucleic acid sequences is also provided inTable 1 (see rows 1 to 7, 9, 11-41 of Column E), Table 3a, and under<223> identifier of the ST25 sequence listing of respective sequence SEQID NOs.

In particularly preferred embodiments, the nucleic acid of the firstaspect comprises at least one coding sequence comprising or consisting aG/C modified coding sequence which is identical or at least 70%, 80%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identical to a codon modified nucleic acid sequence selected fromthe group consisting of SEQ ID NOs: 146, 147, 151, 154, 158, 161, 165,168, 172, 175, 12034-12050, 12052, 12054-12203, 12540-12675,13012-13147, 13519, 13520, 14133-14141, 14151-14159, 14169-14177,23041-23076, 23149-23184, 23261-23296, 23369-23404, 23481-23516,23589-23624, 23701-23736, 23809-23844, 23921-23956, 24029-24064,24141-24176, 24249-24284, 24361-24396, 24469-24504, 24581-24616,24689-24724, 24801-24836, 24909-24944, 25021-25056, 25129-25164,25241-25276, 25349-25384, 25461-25496, 25569-25604, 25681-25716,25789-25824, 25901-25936, 26009-26044, 26121-26156, 26229-26264,26341-26376, 26449-26484, 26561-26596, 26669-26704, 26781-26816,26889-26924 or a fragment or variant of any of these sequences.Additional information regarding each of these suitable nucleic acidsequences encoding may also be derived from the sequence listing, inparticular from the details provided therein under identifier <223>.Suitable coding sequences of the first aspect are provided in Table 1.Further information regarding said nucleic acid sequences is alsoprovided in Table 1 (see rows 1 to 7, 9, 11-41 of Column F), and under<223> identifier of the ST25 sequence listing of respective sequence SEQID NOs.

In even more preferred embodiments, the nucleic acid of the first aspectcomprises at least one coding sequence comprising or consisting a G/Cmodified coding sequence which is identical or at least 70%, 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to a codon modified nucleic acid sequence selected from thegroup consisting of SEQ ID NOs: 136-138, 142, 143, 146, 147, 11731,11798-11801, 11804, 11805, 11808, 11810-11812, 11923, 11953, 12035,12049, 22759-22785, 22965-22982, 23077-23094, 23149 or a fragment orvariant of any of these sequences. Additional information regarding eachof these suitable nucleic acid sequences encoding may also be derivedfrom the sequence listing, in particular from the details providedtherein under identifier <223>. Suitable coding sequences of the firstaspect are provided in Table 1. Further information regarding saidnucleic acid sequences is also provided in Table 1 (see rows 1 to 7, 9,11-41 of Column F), and under <223> identifier of the ST25 sequencelisting of respective sequence SEQ ID NOs.

In a particularly preferred embodiment, the nucleic acid of the firstaspect comprises at least one coding sequence comprising or consisting aG/C modified coding sequence encoding a SARS-CoV-2 antigen which isidentical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a codon modifiednucleic acid sequence according to SEQ ID NOs: 137 or a fragment orvariant thereof.

In further particularly preferred embodiments, the nucleic acid of thefirst aspect comprises at least one coding sequence comprising orconsisting a G/C modified coding sequence encoding a SARS-CoV-2 antigenwhich is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a codonmodified nucleic acid sequence according to SEQ ID NOs: 23090, 23091,23093, 23094 or a fragment or variant thereof.

In further embodiments, the nucleic acid of the first aspect comprisesat least one coding sequence comprising or consisting a coding sequenceencoding a SARS-CoV-2 antigen which is identical or at least 70%, 80%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identical to a codon modified nucleic acid sequence according to SEQID NOs: 23113, 23167 or a fragment or variant thereof.

UTRs:

In preferred embodiments, the nucleic acid of the invention comprises aprotein-coding region (“coding sequence” or “cds”), and 5′-UTR and/or3′-UTR. Notably, UTRs may harbor regulatory sequence elements thatdetermine nucleic acid, e.g. RNA turnover, stability, and localization.Moreover, UTRs may harbor sequence elements that enhance translation. Inmedical application of nucleic acid sequences (including DNA and RNA),translation of the nucleic acid into at least one peptide or protein isof paramount importance to therapeutic efficacy. Certain combinations of3′-UTRs and/or 5′-UTRs may enhance the expression of operably linkedcoding sequences encoding peptides or proteins of the invention. Nucleicacid molecules harboring said UTR combinations advantageously enablerapid and transient expression of antigenic peptides or proteins afteradministration to a subject, preferably after intramuscularadministration. Accordingly, the nucleic acid comprising certaincombinations of 3′-UTRs and/or 5′-UTRs as provided herein isparticularly suitable for administration as a vaccine, in particular,suitable for administration into the muscle, the dermis, or theepidermis of a subject.

Suitably, the nucleic acid of the invention comprises at least oneheterologous 5′-UTR and/or at least one heterologous 3′-UTR. Saidheterologous 5′-UTRs or 3′-UTRs may be derived from naturally occurringgenes or may be synthetically engineered. In preferred embodiments, thenucleic acid, preferably the RNA comprises at least one coding sequenceas defined herein operably linked to at least one (heterologous) 3′-UTRand/or at least one (heterologous) 5′-UTR.

In preferred embodiments, the nucleic acid, e.g. the RNA or DNA,comprises at least one heterologous 3′-UTR.

The term “3′-untranslated region” or “3′-UTR” or “3′-UTR element” willbe recognized and understood by the person of ordinary skill in the art,and are e.g. intended to refer to a part of a nucleic acid moleculelocated 3′ (i.e. downstream) of a coding sequence and which is nottranslated into protein. A 3′-UTR may be part of a nucleic acid, e.g. aDNA or an RNA, located between a coding sequence and an (optional)terminal poly(A) sequence. A 3′-UTR may comprise elements forcontrolling gene expression, also called regulatory elements. Suchregulatory elements may be, e.g., ribosomal binding sites, miRNA bindingsites etc.

Preferably, the nucleic acid comprises a 3′-UTR, which may be derivablefrom a gene that relates to an RNA with enhanced half-life (i.e. thatprovides a stable RNA).

In some embodiments, a 3′-UTR comprises one or more of a polyadenylationsignal, a binding site for proteins that affect a nucleic acid stabilityof location in a cell, or one or more miRNA or binding sites for miRNAs.

MicroRNAs (or miRNA) are 19-25 nucleotide long noncoding RNAs that bindto the 3′-UTR of nucleic acid molecules and down-regulate geneexpression either by reducing nucleic acid molecule stability or byinhibiting translation. E.g., microRNAs are known to regulate RNA, andthereby protein expression, e.g. in liver (miR-122), heart (miR-Id,miR-149), endothelial cells (miR-17-92, miR-126), adipose tissue (let-7,miR-30c), kidney (miR-192, miR-194, miR-204), myeloid cells (miR-142-3p,miR-142-5p, miR-16, miR-21, miR-223, miR-24, miR-27), muscle (miR-133,miR-206, miR-208), and lung epithelial cells (let-7, miR-133, miR-126).The RNA may comprise one or more microRNA target sequences, microRNAsequences, or microRNA seeds. Such sequences may e.g. correspond to anyknown microRNA such as those taught in US2005/0261218 andUS2005/0059005.

Accordingly, miRNA, or binding sites for miRNAs as defined above may beremoved from the 3′-UTR or introduced into the 3′-UTR in order to tailorthe expression of the nucleic acid, e.g. the RNA to desired cell typesor tissues (e.g. muscle cells).

In preferred embodiments, the nucleic acid comprises at least oneheterologous 3′-UTR, wherein the at least one heterologous 3′-UTRcomprises a nucleic acid sequence derived from a 3′-UTR of a geneselected from PSMB3, ALB7, alpha-globin (referred to as “muag”), CASP1,COX6B1, GNAS, NDUFA1 and RPS9, or from a homolog, a fragment or variantof any one of these genes, preferably according to nucleic acidsequences being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ IDNOs: 253-268, 22902-22905, 22892-22895 or a fragment or a variant of anyof these. Particularly preferred nucleic acid sequences in that contextcan be derived from published PCT application WO2019/077001A1, inparticular, claim 9 of WO2019/077001A1. The corresponding 3′-UTRsequences of claim 9 of WO2019/077001A1 are herewith incorporated byreference (e.g., SEQ ID NOs: 23-34 of WO2019/077001A1, or fragments orvariants thereof).

In particularly preferred embodiments, the nucleic acid comprises a3′-UTR derived from an alpha-globin gene. Said 3′-UTR derived from aalpha-globin gene (“muag”) may comprise or consist of a nucleic acidsequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ IDNOs: 267, 268, 22896-22901, 22906-22911 or a fragment or a variantthereof.

In further embodiments, the nucleic acid comprises a 3′-UTR derived froma RPS9 gene. Said 3′-UTR derived from a RPS9 gene may comprise orconsist of a nucleic acid sequence being identical or at least 70%, 80%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identical to SEQ ID NOs: 263 or 264, 22894, 22895, 22904, 22905 or afragment or a variant thereof.

In preferred embodiments, the nucleic acid comprises a 3′-UTR derivedfrom a PSMB3 gene. Said 3′-UTR derived from a PSMB3 gene may comprise orconsist of a nucleic acid sequence being identical or at least 70%, 80%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identical to SEQ ID NOs: 253 or 254, 22892, 22893, 22902, 22903 or afragment or a variant thereof.

In other embodiments, the nucleic acid comprises a 3′-UTR whichcomprises or consists of a nucleic acid sequence being identical or atleast 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identical to SEQ ID NO: 22876-22891 or a fragmentor a variant thereof.

In other embodiments, the nucleic acid may comprise a 3′-UTR asdescribed in WO2016/107877, the disclosure of WO2016/107877 relating to3′-UTR sequences herewith incorporated by reference. Suitable 3′-UTRsare SEQ ID NOs: 1-24 and SEQ ID NOs: 49-318 of WO2016/107877, orfragments or variants of these sequences. In other embodiments, thenucleic acid comprises a 3′-UTR as described in WO2017/036580, thedisclosure of WO2017/036580 relating to 3′-UTR sequences herewithincorporated by reference. Suitable 3′-UTRs are SEQ ID NOs: 152-204 ofWO2017/036580, or fragments or variants of these sequences. In otherembodiments, the nucleic acid comprises a 3′-UTR as described inWO2016/022914, the disclosure of WO2016/022914 relating to 3′-UTRsequences herewith incorporated by reference. Particularly preferred3′-UTRs are nucleic acid sequences according to SEQ ID NOs: 20-36 ofWO2016/022914, or fragments or variants of these sequences.

In preferred embodiments, the nucleic acid, e.g. the RNA or DNA,comprises at least one heterologous 5′-UTR.

The terms “5′-untranslated region” or “5′-UTR” or “5′-UTR element” willbe recognized and understood by the person of ordinary skill in the art,and are e.g. intended to refer to a part of a nucleic acid moleculelocated 5′ (i.e. “upstream”) of a coding sequence and which is nottranslated into protein. A 5′-UTR may be part of a nucleic acid located5′ of the coding sequence. Typically, a 5′-UTR starts with thetranscriptional start site and ends before the start codon of the codingsequence. A 5′-UTR may comprise elements for controlling geneexpression, also called regulatory elements. Such regulatory elementsmay be, e.g., ribosomal binding sites, miRNA binding sites etc. The5′-UTR may be post-transcriptionally modified, e.g. by enzymatic orpost-transcriptional addition of a 5′-cap structure (e.g. for mRNA asdefined below).

Preferably, the nucleic acid comprises a 5′-UTR, which may be derivablefrom a gene that relates to an RNA with enhanced half-life (i.e. thatprovides a stable RNA).

In some embodiments, a 5′-UTR comprises one or more of a binding sitefor proteins that affect an RNA stability or RNA location in a cell, orone or more miRNA or binding sites for miRNAs (as defined above).

Accordingly, miRNA or binding sites for miRNAs as defined above may beremoved from the 5′-UTR or introduced into the 5′-UTR in order to tailorthe expression of the nucleic acid to desired cell types or tissues(e.g. muscle cells).

In preferred embodiments, the nucleic acid comprises at least oneheterologous 5′-UTR, wherein the at least one heterologous 5′-UTRcomprises a nucleic acid sequence derived from a 5′-UTR of gene selectedfrom HSD17B4, RPL32, ASAH1, ATP5A1, MP68, NDUFA4, NOSIP, RPL31, SLC7A3,TUBB4B, and UBQLN2, or from a homolog, a fragment or variant of any oneof these genes according to nucleic acid sequences being identical or atleast 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identical to SEQ ID NOs: 231-252, 22870-22875 or afragment or a variant of any of these. Particularly preferred nucleicacid sequences in that context can be selected from published PCTapplication WO2019/077001A1, in particular, claim 9 of WO2019/077001A1.The corresponding 5′-UTR sequences of claim 9 of WO2019/077001A1 areherewith incorporated by reference (e.g., SEQ ID NOs: 1-20 ofWO2019/077001A1, or fragments or variants thereof).

In preferred embodiments, the nucleic acid comprises a 5′-UTR derivedfrom a RPL31 gene, wherein said 5′-UTR derived from a RPL31 genecomprises or consists of a nucleic acid sequence being identical or atleast 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identical to SEQ ID NOs: 243, 244, 22872, 22873 ora fragment or a variant thereof.

In other embodiments, the nucleic acid comprises a 5′-UTR derived from aSLC7A3 gene, wherein said 5′-UTR derived from a SLC7A3 gene comprises orconsists of a nucleic acid sequence being identical or at least 70%,80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identical to SEQ ID NOs: 245, 246, 22874, 22875 or afragment or a variant thereof.

In particularly preferred embodiments, the nucleic acid comprises a5′-UTR derived from a HSD17B4 gene, wherein said 5′-UTR derived from aHSD17B4 gene comprises or consists of a nucleic acid sequence beingidentical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 231, 232,22870, 22871 or a fragment or a variant thereof.

In other embodiments, the nucleic acid comprises a 5′-UTR whichcomprises or consists of a nucleic acid sequence being identical or atleast 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identical to SEQ ID NO: 22848-22869 or a fragmentor a variant thereof.

In other embodiments, the nucleic acid comprises a 5′-UTR as describedin WO2013/143700, the disclosure of WO2013/143700 relating to 5′-UTRsequences herewith incorporated by reference. Particularly preferred5′-UTRs are nucleic acid sequences derived from SEQ ID NOs: 1-1363, SEQID NO: 1395, SEQ ID NO: 1421 and SEQ ID NO: 1422 of WO2013/143700, orfragments or variants of these sequences. In other embodiments, thenucleic acid comprises a 5′-UTR as described in WO2016/107877, thedisclosure of WO2016/107877 relating to 5′-UTR sequences herewithincorporated by reference. Particularly preferred 5′-UTRs are nucleicacid sequences according to SEQ ID NOs: 25-30 and SEQ ID NOs: 319-382 ofWO2016/107877, or fragments or variants of these sequences. In otherembodiments, the nucleic acid comprises a 5′-UTR as described inWO2017/036580, the disclosure of WO2017/036580 relating to 5′-UTRsequences herewith incorporated by reference. Particularly preferred5′-UTRs are nucleic acid sequences according to SEQ ID NOs: 1-151 ofWO2017/036580, or fragments or variants of these sequences. In otherembodiments, the nucleic acid comprises a 5′-UTR as described inWO2016/022914, the disclosure of WO2016/022914 relating to 5′-UTRsequences herewith incorporated by reference. Particularly preferred5′-UTRs are nucleic acid sequences according to SEQ ID NOs: 3-19 ofWO2016/022914, or fragments or variants of these sequences.

Suitably, in preferred embodiments, the nucleic acid comprises at leastone coding sequence as specified herein encoding at least one antigenicprotein as defined herein, preferably derived from SARS-CoV-2(nCoV-2019) coronavirus, operably linked to a 3′-UTR and/or a 5′-UTRselected from the following 5′UTR/3′UTR combinations (“also referred toUTR designs”):

a-1 (HSD17B4/PSMB3), a-2 (NDUFA4/PSMB3), a-3 (SLC7A3/PSMB3), a-4(NOSIP/PSMB3), a-5 (MP68/PSMB3), b-1 (UBQLN2/RPS9), b-2 (ASAH1/RPS9),b-3 (HSD17B4/RPS9), b-4 (HSD17B4/CASP1), b-5 (NOSIP/COX6B1), c-1(NDUFA4/RPS9), c-2 (NOSIP/NDUFA1), c-3 (NDUFA4/COX6B1), c-4(NDUFA4/NDUFA1), c-5 (ATP5A1/PSMB3), d-1 (RpI31/PSMB3), d-2(ATP5A1/CASP1), d-3 (SLC7A3/GNAS), d-4 (HSD17B4/NDUFA1), d-5(Slc7a3/Ndufa1), e-1 (TUBB4B/RPS9), e-2 (RPL31/RPS9), e-3 (MP68/RPS9),e-4 (NOSIP/RPS9), e-5 (ATP5A1/RPS9), e-6 (ATP5A1/COX6B1), f-1(ATP5A1/GNAS), f-2 (ATP5A1/NDUFA1), f-3 (HSD17B4/COX6B1), f-4(HSD17B4/GNAS), f-5 (MP68/COX6B1), g-1 (MP68/NDUFA1), g-2(NDUFA4/CASP1), g-3 (NDUFA4/GNAS), g-4 (NOSIP/CASP1), g-5 (RPL31/CASP1),h-1 (RPL31/COX6B1), h-2 (RPL31/GNAS), h-3 (RPL31/NDUFA1), h-4(Slc7a3/CASP1), h-5 (SLC7A3/COX6B1), i-1 (SLC7A3/RPS9), i-2(RPL32/ALB7), i-2 (RPL32/ALB7), or i-3 (alpha-globin gene).

In particularly preferred embodiments, the nucleic acid comprises atleast one coding sequence as specified herein encoding at least oneantigenic protein as defined herein, preferably derived from SARS-CoV-2(nCoV-2019) coronavirus, wherein said coding sequence is operably linkedto a HSD17B4 5′-UTR and a PSMB3 3′-UTR (HSD17B4/PSMB3 (UTR design a-1)).

It has been shown by the inventors that this embodiment is particularlybeneficial for induction an immune response against SARS-CoV-2. In thiscontext, it could be shown that already one vaccination was sufficientto result in virus-neutralizing antibody titers.

In further preferred embodiments, the nucleic acid comprises at leastone coding sequence as specified herein encoding at least one antigenicprotein as defined herein, preferably derived from SARS-CoV-2(nCoV-2019) coronavirus, wherein said coding sequence is operably linkedto a SLC7A3 5′-UTR and a PSMB3 3′-UTR (SLC7A3/PSMB3 (UTR design a-3)).

In further preferred embodiments, the nucleic acid comprises at leastone coding sequence as specified herein encoding at least one antigenicprotein as defined herein, preferably derived from SARS-CoV-2(nCoV-2019) coronavirus, wherein said coding sequence is operably linkedto a RPL31 5′-UTR and a RPS9 3′-UTR (RPL31/RPS9 (UTR design e-2)).

In particularly preferred embodiments of the nucleic acid comprises atleast one coding sequence as specified herein encoding at least oneantigenic protein as defined herein, preferably derived from SARS-CoV-2(nCoV-2019) coronavirus, wherein said coding sequence is operably linkedto an alpha-globin (“muag”) 3′-UTR (−/muag)(UTR design i-3)).

In some embodiments, the nucleic acid, e.g. the DNA or RNA may bemonocistronic, bicistronic, or multicistronic.

The term “monocistronic” will be recognized and understood by the personof ordinary skill in the art, and is e.g. intended to refer to a nucleicacid that comprises only one coding sequence. The terms “bicistronic”,or “multicistronic” as used herein will be recognized and understood bythe person of ordinary skill in the art, and are e.g. intended to referto a nucleic acid that may comprise two (bicistronic) or more(multicistronic) coding sequences.

In preferred embodiments, the nucleic acid of the first aspect ismonocistronic.

In other embodiments, the nucleic acid is monocistronic and the codingsequence of said nucleic acid encodes at least two different antigenicpeptides or proteins derived from a SARS-CoV-2 coronavirus. Accordingly,said coding sequence may encode at least two, three, four, five, six,seven, eight and more antigenic peptides or proteins derived from aSARS-CoV-2 coronavirus, linked with or without an amino acid linkersequence, wherein said linker sequence can comprise rigid linkers,flexible linkers, cleavable linkers, or a combination thereof. Suchconstructs are herein referred to as “multi-antigen-constructs”.

In further embodiments, the nucleic acid may be bicistronic ormulticistronic and comprises at least two coding sequences, wherein theat least two coding sequences encode two or more different antigenicpeptides or proteins derived from a SARS-CoV-2 coronavirus. Accordingly,the coding sequences in a bicistronic or multicistronic nucleic acidsuitably encodes distinct antigenic proteins or peptides as definedherein or immunogenic fragments or immunogenic variants thereof.Preferably, the coding sequences in said bicistronic or multicistronicconstructs may be separated by at least one IRES (internal ribosomalentry site) sequence. Thus, the term “encoding two or more antigenicpeptides or proteins” may mean, without being limited thereto, that thebicistronic or multicistronic nucleic acid encodes e.g. at least two,three, four, five, six or more (preferably different) antigenic peptidesor proteins of different SARS-CoV-2 coronavirus isolates. Alternatively,the bicistronic or multicistronic nucleic acid may encode e.g. at leasttwo, three, four, five, six or more (preferably different) antigenicpeptides or proteins derived from the same SARS-CoV-2 coronavirus. Inthat context, suitable IRES sequences may be selected from the list ofnucleic acid sequences according to SEQ ID NOs: 1566-1662 of the patentapplication WO2017/081082, or fragments or variants of these sequences.In this context, the disclosure of WO2017/081082 relating to IRESsequences is herewith incorporated by reference.

It has to be understood that, in the context of the invention, certaincombinations of coding sequences may be generated by any combination ofmonocistronic, bicistronic and multicistronic DNA and/or RNA constructsand/or multi-antigen-constructs to obtain a nucleic acid set encodingmultiple antigenic peptides or proteins as defined herein.

In preferred embodiments, the A/U (A/T) content in the environment ofthe ribosome binding site of the nucleic acid may be increased comparedto the A/U (NT) content in the environment of the ribosome binding siteof its respective wild type or reference nucleic acid. This modification(an increased A/U (A/T) content around the ribosome binding site)increases the efficiency of ribosome binding to the nucleic acid, e.g.to an RNA. An effective binding of the ribosomes to the ribosome bindingsite in turn has the effect of an efficient translation the nucleicacid.

Accordingly, in a particularly preferred embodiment, the nucleic acidcomprises a ribosome binding site, also referred to as “Kozak sequence”identical to or at least 80%, 85%, 90%, 95% identical to any one of thesequences SEQ ID NOs: 180, 181, 22845-22847, or fragments or variantsthereof.

In preferred embodiments, the nucleic acid of the invention comprises atleast one poly(N) sequence, e.g. at least one poly(A) sequence, at leastone poly(U) sequence, at least one poly(C) sequence, or combinationsthereof.

In preferred embodiments, the nucleic acid of the invention, preferablythe RNA comprises at least one poly(A) sequence.

The terms “poly(A) sequence”, “poly(A) tail” or “3′-poly(A) tail” asused herein will be recognized and understood by the person of ordinaryskill in the art, and are e.g. intended to be a sequence of adenosinenucleotides, typically located at the 3′-end of a linear RNA (or in acircular RNA), of up to about 1000 adenosine nucleotides. Preferably,said poly(A) sequence is essentially homopolymeric, e.g. a poly(A)sequence of e.g. 100 adenosine nucleotides has essentially the length of100 nucleotides. In other embodiments, the poly(A) sequence may beinterrupted by at least one nucleotide different from an adenosinenucleotide, e.g. a poly(A) sequence of e.g. 100 adenosine nucleotidesmay have a length of more than 100 nucleotides (comprising 100 adenosinenucleotides and in addition said at least one nucleotide—or a stretch ofnucleotides—different from an adenosine nucleotide). It has to beunderstood that “poly(A) sequence” as defined herein typically relatesto RNA—however in the context of the invention, the term likewiserelates to corresponding sequences in a DNA molecule (e.g. a “poly(T)sequence”).

The poly(A) sequence may comprise about 10 to about 500 adenosinenucleotides, about 10 to about 200 adenosine nucleotides, about 40 toabout 200 adenosine nucleotides, or about 40 to about 150 adenosinenucleotides. Suitably, the length of the poly(A) sequence may be atleast about or even more than about 10, 50, 64, 75, 100, 200, 300, 400,or 500 adenosine nucleotides.

In preferred embodiments, the nucleic acid comprises at least onepoly(A) sequence comprising about 30 to about 200 adenosine nucleotides.In particularly preferred embodiments, the poly(A) sequence comprisesabout 64 adenosine nucleotides (A64). In other particularly preferredembodiments, the poly(A) sequence comprises about 100 adenosinenucleotides (A100). In other embodiments, the poly(A) sequence comprisesabout 150 adenosine nucleotides.

In further embodiments, the nucleic acid comprises at least one poly(A)sequence comprising about 100 adenosine nucleotides, wherein the poly(A)sequence is interrupted by non-adenosine nucleotides, preferably by 10non-adenosine nucleotides (A30-N10-A70).

The poly(A) sequence as defined herein may be located directly at the 3′terminus of the nucleic acid, preferably directly at the 3′ terminus ofan RNA.

In preferred embodiments, the 3′-terminal nucleotide (that is the last3′-terminal nucleotide in the polynucleotide chain) is the 3′-terminal Anucleotide of the at least one poly(A) sequence. The term “directlylocated at the 3′ terminus” has to be understood as being locatedexactly at the 3′ terminus—in other words, the 3′ terminus of thenucleic acid consists of a poly(A) sequence terminating with an Anucleotide.

It has been shown by the inventors that this embodiment is particularlybeneficial for induction an immune response against SARS-CoV-2. In thiscontext, it could be shown that already one vaccination was sufficientto result in virus-neutralizing antibody titers.

In a particularly preferred embodiment the nucleic acid sequence,preferably the RNA comprises a poly(A) sequence of at least 70 adenosinenucleotides, wherein the 3′-terminal nucleotide is an adenosinenucleotide.

In this context it has been shown that ending on an adenosine nucleotidedecreases the induction of IFNalpha by the RNA vaccine. This isparticularly important as the induction of IFNalpha is thought to be themain factor for induction of fever in vaccinated subjects, which ofcourse has to be avoided.

In embodiments where the nucleic acid is an RNA, the poly(A) sequence ofthe nucleic acid is preferably obtained from a DNA template during RNAin vitro transcription. In other embodiments, the poly(A) sequence isobtained in vitro by common methods of chemical synthesis without beingnecessarily transcribed from a DNA template. In other embodiments,poly(A) sequences are generated by enzymatic polyadenylation of the RNA(after RNA in vitro transcription) using commercially availablepolyadenylation kits and corresponding protocols known in the art, oralternatively, by using immobilized poly(A)polymerases e.g. using amethods and means as described in WO2016/174271.

The nucleic acid may comprise a poly(A) sequence obtained by enzymaticpolyadenylation, wherein the majority of nucleic acid molecules compriseabout 100 (+/−20) to about 500 (+/−50), preferably about 250 (+/−20)adenosine nucleotides.

In other embodiments, the nucleic acid may comprise a poly(A) sequencederived from a template DNA and may additionally comprise at least oneadditional poly(A) sequence generated by enzymatic polyadenylation, e.g.as described in WO2016/091391.

In further embodiments, the nucleic acid comprises at least onepolyadenylation signal.

In other embodiments, the nucleic acid may comprise at least one poly(C)sequence.

The term “poly(C) sequence” as used herein is intended to be a sequenceof cytosine nucleotides of up to about 200 cytosine nucleotides. Inpreferred embodiments, the poly(C) sequence comprises about 10 to about200 cytosine nucleotides, about 10 to about 100 cytosine nucleotides,about 20 to about 70 cytosine nucleotides, about 20 to about 60 cytosinenucleotides, or about 10 to about 40 cytosine nucleotides. In aparticularly preferred embodiment, the poly(C) sequence comprises about30 cytosine nucleotides.

In preferred embodiments, the nucleic acid of the invention comprises atleast one histone stem-loop (hSL).

The term “histone stem-loop” (abbreviated as “hSL” in e.g. the sequencelisting) is intended to refer to nucleic acid sequences that form astem-loop secondary structure predominantly found in histone mRNAs.

Histone stem-loop sequences/structures may suitably be selected fromhistone stem-loop sequences as disclosed in WO2012/019780, thedisclosure relating to histone stem-loop sequences/histone stem-loopstructures incorporated herewith by reference. A histone stem-loopsequence that may be used within the present invention may preferably bederived from formulae (I) or (II) of WO2012/019780. According to afurther preferred embodiment, the nucleic acid may comprise at least onehistone stem-loop sequence derived from at least one of the specificformulae (Ia) or (IIa) of the patent application WO2012/019780.

In preferred embodiments, the nucleic acid of the invention comprises atleast one histone stem-loop, wherein said histone stem-loop (hSL)comprises or consists a nucleic acid sequence identical or at least 70%,80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical toSEQ ID NOs: 178 or 179, or fragments or variants thereof.

In other embodiments, the RNA of the first aspect does not comprise ahistone stem-loop as defined herein.

In some embodiments, in particular in embodiments that relate to RNA,the nucleic acid comprises a 3′-terminal sequence element. Said3′-terminal sequence element comprises a poly(A) sequence and optionallya histone-stem-loop sequence. Accordingly, the nucleic acid of theinvention comprises at least one 3′-terminal sequence element comprisingor consisting of a nucleic acid sequence being identical or at least70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identicalto SEQ ID NOs: 182-230, 22912, 22913 or a fragment or variant thereof.

In preferred embodiments, in particular in embodiments that relate toRNA, the nucleic acid comprises a 3′-terminal sequence element. Said3′-terminal sequence element comprises a poly(A) sequence and optionallya histone-stem-loop sequence. Accordingly, the nucleic acid of theinvention comprises at least one 3′-terminal sequence element comprisingor consisting of a nucleic acid sequence being identical or at least70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identicalto SEQ ID NOs: 182, 187, 189, 192, 199, 207, or a fragment or variantthereof.

In various embodiments, in particular in embodiments that relate to RNA,the nucleic acid may comprise a 5′-terminal sequence element accordingto SEQ ID NOs: 176 or 177, 22840-22844, or a fragment or variantthereof. Such a 5′-terminal sequence element comprises e.g. a bindingsite for T7 RNA polymerase. Further, the first nucleotide of said5′-terminal start sequence may preferably comprise a 2′ methylation,e.g. 2′ methylated guanosine or a 2′ methylated adenosine.

Preferably, the nucleic acid of the first aspect, e.g. the RNA or DNA,typically comprises about 50 to about 20000 nucleotides, or about 500 toabout 10000 nucleotides, or about 1000 to about 10000 nucleotides, orpreferably about 1000 to about 5000 nucleotides, or even more preferablyabout 2000 to about 5000 nucleotides.

In some embodiments, the nucleic acid is a DNA or an RNA.

In various embodiments, the DNA is a plasmid DNA or a linear coding DNAconstruct, wherein the DNA comprises or consists of the nucleic acidelements as defined herein (e.g. including coding sequences, UTRs,poly(A/T), polyadenylation signal, a promoter).

In preferred embodiments, the nucleic acid is a DNA expression vector.Such a DNA expression vector may be selected from the group consistingof a bacterial plasmid, an adenovirus, a poxvirus, a parapoxivirus (orfvirus), a vaccinia virus, a fowlpox virus, a herpes virus, anadeno-associated virus (AAV), an alphavirus, a lentivirus, a lambdaphage, a lymphocytic choriomeningitis virus and a Listeria sp,Salmonella sp.

Suitably, the DNA may also comprise a promoter that is operably linkedto the SARS-CoV-2 antigen coding sequence. The promoter operably linkedto the antigen coding sequence can be e.g. a promoter from simian virus40 (SV40), a mouse mammary tumor virus (MMTV) promoter, a humanimmunodeficiency virus (HIV) promoter such as the bovineimmunodeficiency virus (BIV) long terminal repeat (LTR) promoter, aMoloney virus promoter, an avian leukosis virus (ALV) promoter, acytomegalovirus (CMV) promoter such as the CMV immediate early promoter,Epstein Barr virus (EBV) promoter, or a Rous sarcoma virus (RSV)promoter. The promoter can also be a promoter from a human gene such ashuman actin, human myosin, human hemoglobin, human muscle creatine, orhuman metalothionein. The promoter can also be a tissue specificpromoter, such as a muscle or skin specific promoter, natural orsynthetic. Examples of such promoters are described in US patentapplication publication no. US20040175727. In preferred embodiments, thevector can be pVAX, pcDNA3.0, or provax, or any other expression vectorcapable of expressing DNA encoding the coronavirus antigen and enablinga cell to translate the sequence to an antigen that is recognized by theimmune system.

Further suitable plasmid DNA may be generated to allow efficientproduction of the encoded SARS-CoV-2 antigen in cell lines, e.g. ininsect cell lines, for example using vectors as described inWO2009150222A2 and as defined in PCT claims 1 to 33, the disclosurerelating to claims 1 to 33 of WO2009150222A2 herewith incorporated byreference.

In other embodiments, the nucleic acid of the first aspect is anadenovirus based vector. Such an adenovirus based vector may comprise atleast one coding sequence encoding at least one SARS-CoV-2 antigenicpeptide or protein as defined herein.

In the context of the invention, any suitable adenovirus based vectormay be used such as those described in WO2005/071093 or WO2006/048215.Suitably, the adenovirus based vector used is a simian adenovirus,thereby avoiding dampening of the immune response after vaccination bypre-existing antibodies to common human entities such as AdHu5. Suitablesimian adenovirus vectors include AdCh63 (patent number WO/2005/071093)or AdCh68 (Cohen et al J. Gen Virol 2002 83:151) but others may also beused. Suitably the adenovirus vector will have the E1 region deleted,rendering it replication-deficient in human cells. Other regions of theadenovirus such as E3 and E4 may also be deleted.

In additional embodiments, the nucleic acid of the first aspect is anorf virus based vector. Such an adenovirus based vector may comprise atleast one coding sequence encoding at least one SARS-CoV-2 antigenicpeptide or protein as defined herein.

In particularly preferred embodiments of the invention, the nucleic acidof the invention is an RNA.

Preferably, the RNA typically comprises about 50 to about 20000nucleotides, or about 500 to about 10000 nucleotides, or about 1000 toabout 10000 nucleotides, or preferably about 1000 to about 5000nucleotides, or even more preferably about 2000 to about 5000nucleotides.

According to preferred embodiments, the nucleic acid is an RNA,preferably a coding RNA.

In preferred embodiments, the coding RNA may be selected from an mRNA, a(coding) self-replicating RNA, a (coding) circular RNA, a (coding) viralRNA, or a (coding) replicon RNA.

In other embodiments, the coding RNA is a circular RNA. As used herein,“circular RNA” or “circRNAs” have to be understood as a circularpolynucleotide constructs that encode at least one antigenic peptide orprotein as defined herein. Preferably, such a circRNA is a singlestranded RNA molecule. In preferred embodiments, said circRNA comprisesat least one coding sequence encoding at least one antigenic proteinfrom a SARS-CoV-2 coronavirus, or an immunogenic fragment or animmunogenic variant thereof.

In further embodiments, the coding RNA is a replicon RNA. The term“replicon RNA” will be recognized and understood by the person ofordinary skill in the art, and is e.g. intended to be an optimizedself-replicating RNA. Such constructs may include replicase elementsderived from e.g. alphaviruses (e.g. SFV, SIN, VEE, or RRV) and thesubstitution of the structural virus proteins with the nucleic acid ofinterest (that is, the coding sequence encoding an antigenic peptide orprotein of a SARS-CoV-2 coronavirus). Alternatively, the replicase maybe provided on an independent coding RNA construct or a coding DNAconstruct. Downstream of the replicase may be a sub-genomic promoterthat controls replication of the replicon RNA.

In particularly preferred embodiments, the at least one nucleic acid isnot a replicon RNA or a self-replicating RNA. In particularly preferredembodiments, the nucleic acid of the invention is an mRNA.

Preferably, the mRNA does not comprise a replicase element (e.g. anucleic acid encoding a replicase).

The terms “RNA” and “mRNA” will be recognized and understood by theperson of ordinary skill in the art, and are e.g. intended to be aribonucleic acid molecule, i.e. a polymer consisting of nucleotides.These nucleotides are usually adenosine-monophosphate,uridine-monophosphate, guanosine-monophosphate andcytidine-monophosphate monomers which are connected to each other alonga so-called backbone. The backbone is formed by phosphodiester bondsbetween the sugar, i.e. ribose, of a first and a phosphate moiety of asecond, adjacent monomer. The specific succession of the monomers iscalled the RNA-sequence. The mRNA (messenger RNA) provides thenucleotide coding sequence that may be translated into an amino-acidsequence of a particular peptide or protein.

In the context of the invention, the coding RNA, preferably the mRNA,may provide at least one coding sequence encoding an antigenic proteinfrom a SARS-CoV-2 that is translated into a (functional) antigen afteradministration (e.g. after administration to a subject, e.g. a humansubject).

Accordingly, the coding RNA, preferably the mRNA, is suitable for avaccine, preferably a SARS-CoV-2 vaccine.

Suitably, the coding RNA may be modified by the addition of a 5′-capstructure, which preferably stabilizes the coding RNA and/or enhancesexpression of the encoded antigen and/or reduces the stimulation of theinnate immune system (after administration to a subject). A 5′-capstructure is of particular importance in embodiments where the nucleicacid is an RNA, in particular a linear coding RNA, e.g. a linear mRNA ora linear coding replicon RNA.

Accordingly, in preferred embodiments, the RNA, in particular the codingRNA comprises a 5′-cap structure, preferably cap0, cap1, cap2, amodified cap0, or a modified cap1 structure.

The term “5′-cap structure” as used herein will be recognized andunderstood by the person of ordinary skill in the art, and is e.g.intended to refer to a 5′ modified nucleotide, particularly a guaninenucleotide, positioned at the 5′-end of an RNA, e.g. an mRNA.Preferably, the 5′-cap structure is connected via a 5′-5′-triphosphatelinkage to the RNA.

5′-cap structures which may be suitable in the context of the presentinvention are cap0 (methylation of the first nucleobase, e.g. m7GpppN),cap1 (additional methylation of the ribose of the adjacent nucleotide ofm7GpppN), cap2 (additional methylation of the ribose of the 2ndnucleotide downstream of the m7GpppN), cap3 (additional methylation ofthe ribose of the 3rd nucleotide downstream of the m7GpppN), cap4(additional methylation of the ribose of the 4th nucleotide downstreamof the m7GpppN), ARCA (anti-reverse cap analogue), modified ARCA (e.g.phosphothioate modified ARCA), inosine, N1-methyl-guanosine,2′-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine,2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine.

A 5′-cap (cap0 or cap1) structure may be formed in chemical RNAsynthesis or in RNA in vitro transcription (co-transcriptional capping)using cap analogues.

The term “cap analogue” as used herein will be recognized and understoodby the person of ordinary skill in the art, and is e.g. intended torefer to a non-polymerizable di-nucleotide or tri-nucleotide that hascap functionality in that it facilitates translation or localization,and/or prevents degradation of a nucleic acid molecule, particularly ofan RNA molecule, when incorporated at the 5′-end of the nucleic acidmolecule. Non-polymerizable means that the cap analogue will beincorporated only at the 5′-terminus because it does not have a 5′triphosphate and therefore cannot be extended in the 3′-direction by atemplate-dependent polymerase, particularly, by template-dependent RNApolymerase. Examples of cap analogues include, but are not limited to, achemical structure selected from the group consisting of m7GpppG,m7GpppA, m7GpppC; unmethylated cap analogues (e.g. GpppG); dimethylatedcap analogue (e.g. m2,7GpppG), trimethylated cap analogue (e.g.m2,2,7GpppG), dimethylated symmetrical cap analogues (e.g. m7Gpppm7G),or anti reverse cap analogues (e.g. ARCA; m7,2′OmeGpppG, m7,2′dGpppG,m7,3′OmeGpppG, m7,3′dGpppG and their tetraphosphate derivatives).Further cap analogues have been described previously (WO2008/016473,WO2008/157688, WO2009/149253, WO2011/015347, and WO2013/059475). Furthersuitable cap analogues in that context are described in WO2017/066793,WO2017/066781, WO2017/066791, WO2017/066789, WO2017/053297,WO2017/066782, WO2018/075827 and WO2017/066797 wherein the disclosuresreferring to cap analogues are incorporated herewith by reference.

In some embodiments, a modified cap1 structure is generated usingtri-nucleotide cap analogue as disclosed in WO2017/053297,WO2017/066793, WO2017/066781, WO2017/066791, WO2017/066789,WO2017/066782, WO2018/075827 and WO2017/066797. In particular, any capstructures derivable from the structure disclosed in claim 1-5 ofWO2017/053297 may be suitably used to co-transcriptionally generate amodified cap1 structure. Further, any cap structures derivable from thestructure defined in claim 1 or claim 21 of WO2018/075827 may besuitably used to co-transcriptionally generate a modified cap1structure.

In preferred embodiments, the (coding) RNA, in particular the mRNAcomprises a cap1 structure.

In preferred embodiments, the 5′-cap structure may suitably be addedco-transcriptionally using tri-nucleotide cap analogue as defined hereinin an RNA in vitro transcription reaction as defined herein.

In preferred embodiments, the cap1 structure of the coding RNA of theinvention is formed using co-transcriptional capping usingtri-nucleotide cap analogues m7G(5′)ppp(5′)(2′OMeA)pG orm7G(5′)ppp(5′)(2′OMeG)pG. A preferred cap1 analogues in that context ism7G(5′)ppp(5′)(2′OMeA)pG.

In other preferred embodiments, the cap1 structure of the RNA of theinvention is formed using co-transcriptional capping usingtri-nucleotide cap analogue 3′OMe-m7G(5′)ppp(5′)(2′OMeA)pG.

In other embodiments, a cap0 structure of the RNA of the invention isformed using co-transcriptional capping using cap analogue3′OMe-m7G(5′)ppp(5′)G.

In other embodiments, the 5′-cap structure is formed via enzymaticcapping using capping enzymes (e.g. vaccinia virus capping enzymesand/or cap-dependent 2′-0 methyltransferases) to generate cap0 or cap1or cap2 structures.

The 5′-cap structure (cap0 or cap1) may be added using immobilizedcapping enzymes and/or cap-dependent 2′-0 methyltransferases usingmethods and means disclosed in WO2016/193226.

In preferred embodiments, about 70%, 75%, 80%, 85%, 90%, 95% of the RNA(species) comprises a cap1 structure as determined using a cappingassay. In preferred embodiments, less than about 20%, 15%, 10%, 5%, 4%,3%, 2%, 1% of the RNA (species) does not comprises a cap1 structure asdetermined using a capping assay. In other preferred embodiments, about70%, 75%, 80%, 85%, 90%, 95% of the RNA (species) comprises a cap0structure as determined using a capping assay. In preferred embodiments,less than about 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1% of the RNA (species)does not comprises a cap0 structure as determined using a capping assay.

The term “RNA species” is not restricted to mean “one single molecule”but is understood to comprise an ensemble of essentially identical RNAmolecules. Accordingly, it may relate to a plurality of essentiallyidentical (coding) RNA molecules.

For determining the presence/absence of a cap0 or a cap1 structure, acapping assays as described in published PCT application WO2015/101416,in particular, as described in claims 27 to 46 of published PCTapplication WO2015/101416 can be used. Other capping assays that may beused to determine the presence/absence of a cap0 or a cap1 structure ofan RNA are described in PCT/EP2018/08667, or published PCT applicationsWO2014/152673 and WO2014/152659.

In preferred embodiments, the RNA comprises an m7G(5′)ppp(5′)(2′OMeA)cap structure. In such embodiments, the coding RNA comprises a5′-terminal m7G cap, and an additional methylation of the ribose of theadjacent nucleotide of m7GpppN, in that case, a 2′ methylated Adenosine.Preferably, about 70%, 75%, 80%, 85%, 90%, 95% of the RNA (species)comprises such a cap1 structure as determined using a capping assay.

In other preferred embodiments, the RNA comprises anm7G(5′)ppp(5′)(2′OMeG) cap structure. In such embodiments, the codingRNA comprises a 5′-terminal m7G cap, and an additional methylation ofthe ribose of the adjacent nucleotide, in that case, a 2′ methylatedguanosine. Preferably, about 70%, 75%, 80%, 85%, 90%, 95% of the codingRNA (species) comprises such a cap1 structure as determined using acapping assay.

Accordingly, the first nucleotide of said RNA or mRNA sequence, that is,the nucleotide downstream of the m7G(5′)ppp structure, may be a 2′methylated guanosine or a 2′ methylated adenosine.

According to some embodiments, the RNA is a modified RNA, wherein themodification refers to chemical modifications comprising backbonemodifications as well as sugar modifications or base modifications.

A modified RNA may comprise nucleotide analogues/modifications, e.g.backbone modifications, sugar modifications or base modifications. Abackbone modification in the context of the invention is a modification,in which phosphates of the backbone of the nucleotides of the RNA arechemically modified. A sugar modification in the context of theinvention is a chemical modification of the sugar of the nucleotides ofthe RNA. Furthermore, a base modification in the context of theinvention is a chemical modification of the base moiety of thenucleotides of the RNA. In this context, nucleotide analogues ormodifications are preferably selected from nucleotide analogues whichare applicable for transcription and/or translation.

In particularly preferred embodiments, the nucleotideanalogues/modifications which may be incorporated into a modified RNA asdescribed herein are preferably selected from2-amino-6-chloropurineriboside-5′-triphosphate,2-Aminopurine-riboside-5′-triphosphate;2-aminoadenosine-5′-triphosphate,2′-Amino-2′-deoxycytidine-triphosphate, 2-thiocytidine-5′-triphosphate,2-thiouridine-5′-triphosphate, 2′-Fluorothymidine-5′-triphosphate,2′-O-Methyl-inosine-5′-triphosphate 4-thiouridine-5′-triphosphate,5-aminoallylcytidine-5′-triphosphate,5-aminoallyluridine-5′-triphosphate, 5-bromocytidine-5′-triphosphate,5-bromouridine-5′-triphosphate,5-Bromo-2′-deoxycytidine-5′-triphosphate,5-Bromo-2′-deoxyuridine-5′-triphosphate, 5-iodocytidine-5′-triphosphate,5-lodo-2′-deoxycytidine-5′-triphosphate, 5-iodouridine-5′-triphosphate,5-lodo-2′-deoxyuridine-5′-triphosphate,5-methylcytidine-5′-triphosphate, 5-methyluridine-5′-triphosphate,5-Propynyl-2′-deoxycytidine-5′-triphosphate,5-Propynyl-2′-deoxyuridine-5′-triphosphate,6-azacytidine-5′-triphosphate, 6-azauridine-5′-triphosphate,6-chloropurineriboside-5′-triphosphate,7-deazaadenosine-5′-triphosphate, 7-deazaguanosine-5′-triphosphate,8-azaadenosine-5′-triphosphate, 8-azidoadenosine-5′-triphosphate,benzimidazole-riboside-5′-triphosphate,N1-methyladenosine-5′-triphosphate, N1-methylguanosine-5′-triphosphate,N6-methyladenosine-5′-triphosphate, 06-methylguanosine-5′-triphosphate,pseudouridine-5′-triphosphate, or puromycin-5′-triphosphate,xanthosine-5′-triphosphate. Particular preference is given tonucleotides for base modifications selected from the group ofbase-modified nucleotides consisting of5-methylcytidine-5′-triphosphate, 7-deazaguanosine-5′-triphosphate,5-bromocytidine-5′-triphosphate, and pseudouridine-5′-triphosphate,pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine,2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine,5-hydroxyuridine, 3-methyluridine, 5-carboxymethyl-uridine,1-carboxymethyl-pseudouridine, 5-propynyl-uridine,1-propynyl-pseudouridine, 5-taurinomethyluridine,1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine,1-taurinomethyl-4-thio-uridine, 5-methyl-uridine,1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine,2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine,2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine,dihydropseudouridine, 2-thio-dihydrouridine,2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine,4-methoxy-pseudouridine, and 4-methoxy-2-thio-pseudouridine,5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine,5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine,1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine,2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine,4-thio-1-methyl-pseudoisocytidine,4-thio-1-methyl-1-deaza-pseudoisocytidine,1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine,5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine,2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine,4-methoxy-pseudoisocytidine, and 4-methoxy-1-methyl-pseudoisocytidine,2-aminopurine, 2,6-diaminopurine, 7-deaza-adenine,7-deaza-8-aza-adenine, 7-deaza-2-aminopurine,7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine,7-deaza-8-aza-2,6-diaminopurine, 1-methyladenosine, N6-methyladenosine,N6-isopentenyladenosine, N6-(cis-hydroxyisopentenyl)adenosine,2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine,N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine,2-methylthio-N6-threonyl carbamoyladenosine, N6,N6-dimethyladenosine,7-methyladenine, 2-methylthio-adenine, and 2-methoxy-adenine, inosine,1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine,7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine,6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine,6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine,1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine,8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine,N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine,5′-O-(1-thiophosphate)-adenosine, 5′-O-(1-thiophosphate)-cytidine,5′-O-(1-thiophosphate)-guanosine, 5′-O-(1-thiophosphate)-uridine,5′-O-(1-thiophosphate)-pseudouridine, 6-aza-cytidine, 2-thio-cytidine,alpha-thio-cytidine, Pseudo-iso-cytidine, 5-aminoallyl-uridine,5-iodo-uridine, N1-methyl-pseudouridine, 5,6-dihydrouridine,alpha-thio-uridine, 4-thio-uridine, 6-aza-uridine, 5-hydroxy-uridine,deoxy-thymidine, 5-methyl-uridine, Pyrrolo-cytidine, inosine,alpha-thio-guanosine, 6-methyl-guanosine, 5-methyl-cytdine,8-oxo-guanosine, 7-deaza-guanosine, N1-methyl-adenosine,2-amino-6-Chloro-purine, N6-methyl-2-amino-purine, Pseudo-iso-cytidine,6-Chloro-purine, N6-methyl-adenosine, alpha-thio-adenosine,8-azido-adenosine, 7-deazaadenosine.

In some embodiments, the at least one modified nucleotide is selectedfrom pseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine,2-thiouridine, 4′-thiouridine, 5-methylcytosine, 5-methyluridine,2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine,2-thio-5-aza-uridine, 2-thio-dihydropseudouridine,2-thio-dihydrouridine, 2-thio-pseudouridine,4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine,4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine,dihydropseudouridine, 5-methoxyuridine and 2′-O-methyl uridine.

In some embodiments, 100% of the uracil in the coding sequence asdefined herein have a chemical modification, preferably a chemicalmodification is in the 5-position of the uracil.

Particularly preferred in the context of the invention are pseudouridine(ψ), N1-methylpseudouridine (m1ψ), 5-methylcytosine, and5-methoxyuridine.

In some embodiments, however, a polynucleotide molecule of theembodiments does not include any N1-methylpseudouridine (m1ψ)substituted positions. In further aspects, a polynucleotide molecule ofthe embodiments does not include any pseudouridine (ψ),N1-methylpseudouridine (m1ψ), 5-methylcytosine, and 5-methoxyuridinesubstituted position. In still further aspects, a polynucleotidemolecule of the embodiments comprises a coding sequence that consistsonly of G, C, A and U nucleotides.

Incorporating modified nucleotides such as pseudouridine (ψ),N1-methylpseudouridine (m1ψ), 5-methylcytosine, and/or 5-methoxyuridineinto the coding sequence of the RNA may be advantageous as unwantedinnate immune responses (upon administration of the coding RNA or thevaccine) may be adjusted or reduced (if required).

In some embodiments, the RNA comprises at least one coding sequenceencoding a SARS-CoV-2 antigenic protein as defined herein, wherein saidcoding sequence comprises at least one modified nucleotide selected frompseudouridine (ψ) and N1-methylpseudouridine (m1ψ), preferably whereinall uracil nucleotides are replaced by pseudouridine (ψ) nucleotidesand/or N1-methylpseudouridine (m1ψ) nucleotides.

In preferred embodiments, the RNA does not compriseN1-methylpseudouridine (m1ψ) substituted positions. In furtherembodiments, the RNA does not comprise pseudouridine (ψ),N1-methylpseudouridine (m1ψ), 5-methylcytosine, and 5-methoxyuridinesubstituted position.

In preferred embodiments, the RNA comprises a coding sequence thatconsists only of G, C, A and U nucleotides and therefore does notcomprise modified nucleotides (except of the Cap analogue)

Nucleic Acid, Preferably mRNA Constructs Suitable for a CoronavirusVaccine:

In various embodiments the nucleic acid, preferably the mRNA comprises,preferably in 5′- to 3′-direction, the following elements:

-   A) 5′-cap structure, preferably as specified herein;-   B) 5′-terminal start element, preferably as specified herein;-   C) optionally, a 5′-UTR, preferably as specified herein;-   D) a ribosome binding site, preferably as specified herein;-   E) at least one coding sequence, preferably as specified herein;-   F) 3′-UTR, preferably as specified herein;-   G) optionally, poly(A) sequence, preferably as specified herein;-   H) optionally, poly(C) sequence, preferably as specified herein;-   I) optionally, histone stem-loop preferably as specified herein;-   J) optionally, 3′-terminal sequence element, preferably as specified    herein.

In preferred embodiments the nucleic acid, preferably the mRNA,comprises the following elements preferably in 5′- to 3′-direction:

-   A) 5′-cap structure selected from m7G(5′), m7G(5′)ppp(5′)(2′OMeA),    or m7G(5′)ppp(5′)(2′OMeG);-   B) 5′-terminal start element selected from SEQ ID NOs: 176 or 177 or    fragments or variants thereof;-   C) optionally, a 5′-UTR derived from a HSD17B4 gene;-   D) a ribosome binding site selected from SEQ ID NOs: 180, 181,    22845-22847 or fragments or variants thereof;-   E) at least one coding sequence selected from SEQ ID NOs: 116-132,    134-138, 140-143, 145-175, 11664-11813, 11815, 11817-12050, 12052,    12054-12203, 13514, 13515, 13519, 13520, 14124-14141, 22759,    22764-22785, 22969-23184 or fragments or variants thereof;-   F) 3′-UTR derived from a 3′-UTR of a PSMB3 gene or an alpha-globin    gene (“muag”);-   G) optionally, poly(A) sequence comprising about 30 to about 500    adenosines;-   H) optionally, poly(C) sequence comprising about 10 to about 100    cytosines;-   I) optionally, histone stem-loop selected from SEQ ID NOs: 178 or    179;-   J) optionally, 3′-terminal sequence element selected from SEQ ID    NOs: 182-230.

In particularly preferred embodiments the nucleic acid, preferably themRNA, comprises the following elements in 5′- to 3′-direction:

-   A) cap1 structure as defined herein;-   B) coding sequence selected from SEQ ID NOs: 116-132, 134-138,    140-143, 145-175, 11664-11813, 11815, 11817-12050, 12052,    12054-12203, 13514, 13515, 13519, 13520, 14124-14141, 22759,    22764-22785, 22969-23184 or fragments or variants thereof;-   C) 3′-UTR derived from a 3′-UTR of a muag gene as defined herein,    preferably according to SEQ ID NO: 267 or 268, 22896-22901,    22906-22911;-   D) poly(A) sequence comprising about 64 A nucleotides.-   E) poly(C) sequence comprising about 10 to about 100 cytosines;-   F) histone stem-loop selected from SEQ ID NOs: 178 or 179;

In preferred embodiments the nucleic acid, preferably the mRNA,comprises the following elements in 5′- to 3′-direction:

-   A) cap1 structure as defined herein;-   B) 5′-UTR derived from a HSD17B4 gene as defined herein, preferably    according to SEQ ID NO: 231 or 232;-   C) coding sequence selected from SEQ ID NOs: 116-132, 134-138,    140-143, 145-175, 11664-11813, 11815, 11817-12050, 12052,    12054-12203, 13514, 13515, 13519, 13520, 14124-14141, 22759,    22764-22785, 22969-23184 or fragments or variants thereof;-   D) 3′-UTR derived from a 3′-UTR of a PSMB3 gene as defined herein,    preferably according to SEQ ID NO: 253 or 254;-   E) poly(A) sequence comprising about 64 A nucleotides.-   F) optionally a poly(C) sequence comprising about 10 to about 100    cytosines;-   G) histone stem-loop selected from SEQ ID NOs: 178 or 179;-   H) optionally, 3′-terminal sequence element SEQ ID NOs: 182-230.

In particularly preferred embodiments the nucleic acid, preferably themRNA, comprises the following elements in 5′- to 3′-direction:

-   A) cap1 structure as defined herein;-   B) 5′-UTR derived from a HSD17B4 gene as defined herein, preferably    according to SEQ ID NO: 231 or 232;-   C) coding sequence selected from SEQ ID NOs: 116-132, 134-138,    140-143, 145-175, 11664-11813, 11815, 11817-12050, 12052,    12054-12203, 13514, 13515, 13519, 13520, 14124-14141, 22759,    22764-22785, 22969-23184 or fragments or variants thereof;-   D) 3′-UTR derived from a 3′-UTR of a PSMB3 gene as defined herein,    preferably according to SEQ ID NO: 253 or 254;-   E) a histone stem-loop selected from SEQ ID NOs: 178 or 179;-   F) poly(A) sequence comprising about 100 A nucleotides, preferably    representing the 3′ terminus.

In further preferred embodiments the nucleic acid, preferably the mRNA,comprises the following elements in 5′- to 3′-direction:

-   A) cap1 structure as defined herein;-   B) 5′-UTR derived from a HSD17B4 gene as defined herein, preferably    according to SEQ ID NO: 231 or 232; C) coding sequence selected from    SEQ ID NOs: 116-132, 134-138, 140-143, 145-175, 11664-11813, 11815,    11817-12050, 12052, 12054-12203, 13514, 13515, 13519, 13520,    14124-14141, 22759, 22764-22785, 22969-23184 or fragments or    variants thereof;-   D) 3′-UTR derived from a 3′-UTR of a PSMB3 gene as defined herein,    preferably according to SEQ ID NO: 253 or 254;-   F) poly(A) sequence comprising about 100 A nucleotides, preferably    representing the 3′ terminus.

In further preferred embodiments the nucleic acid, preferably the mRNA,comprises the following elements in 5′- to 3′-direction:

-   A) cap1 structure as defined herein;-   B) 5′-UTR derived from a SLC7A3 gene as defined herein, preferably    according to SEQ ID NO: 245 or 246;-   C) coding sequence selected from SEQ ID NOs: 116-132, 134-138,    140-143, 145-175, 11664-11813, 11815, 11817-12050, 12052,    12054-12203, 13514, 13515, 13519, 13520, 14124-14141, 22759,    22764-22785, 22969-23184 or fragments or variants thereof;-   D) 3′-UTR derived from a 3′-UTR of a PSMB3 gene as defined herein,    preferably according to SEQ ID NO: 253 or 254;-   E) optionally a histone stem-loop selected from SEQ ID NOs: 178 or    179;-   F) poly(A) sequence comprising about 100 A nucleotides, preferably    representing the 3′ terminus.

In further preferred embodiments the nucleic acid, preferably the mRNA,comprises the following elements in 5′- to 3′-direction:

-   A) cap1 structure as defined herein;-   B) 5′-UTR derived from a RPL31 gene as defined herein, preferably    according to SEQ ID NO: 243 or 243;-   C) coding sequence selected from SEQ ID NOs: 116-132, 134-138,    140-143, 145-175, 11664-11813, 11815, 11817-12050, 12052,    12054-12203, 13514, 13515, 13519, 13520, 14124-14141, 22759,    22764-22785, 22969-23184 or fragments or variants thereof;-   D) 3′-UTR derived from a 3′-UTR of a RPS9 gene as defined herein,    preferably according to SEQ ID NO: 263 or 264;-   E) optionally a histone stem-loop selected from SEQ ID NOs: 178 or    179;-   F) poly(A) sequence comprising about 100 A nucleotides, preferably    representing the 3′ terminus.

Preferred nucleic acid sequences, preferably mRNA sequences of theinvention are provided in Table 3a. Therein, each row represents aspecific suitable SARS-CoV-2 (nCoV-2019) construct of the invention(compare with Table 1), wherein the description of the SARS-CoV-2construct is indicated in column A of Table 3a and the SEQ ID NOs of theamino acid sequence of the respective SARS-CoV-2 construct is providedin column B. The corresponding SEQ ID NOs of the coding sequencesencoding the respective SARS-CoV-2 constructs are provided in inTable 1. Further information is provided under <223> identifier of therespective SEQ ID NOs in the sequence listing.

The corresponding nucleic acid, preferably coding RNA sequences, inparticular mRNA sequences comprising preferred coding sequences areprovided in columns C and D, wherein column C provides nucleic acidsequences with an UTR combination “HSD17B4/PSMB3” as defined herein,wherein column D provides nucleic acid sequences with an “alpha-globin”3′ UTR as defined herein.

TABLE 3a Nucleic acid, preferably mRNA constructs suitable for acoronavirus vaccine row A B C D 1 Full-length spike protein; S 1-9,274-340, 148, 155,12204-12337, 162, 169, 12676- 22737, 22739,12473-12540, 22791, 12809, 12945-13012, 22741, 22743, 22793, 22795,22797, 22818, 22820, 22822, 22745, 22747, 22799, 22801, 22803, 22824,22826, 22828, 22749, 22751, 22805, 22807, 22809, 22830, 22832, 22834,22753, 22755, 22811, 23409-23516 22836, 22838, 23189- 22757, 22929-23296 22946 2 Stabilized spike protein; S_stab_PP 10-18, 341-407,149-151, 156-158, 163-165, 170-172, 22738, 22740, 12338, 12541, 22792,12810, 13013, 22819, 22742, 22744, 22794, 22796, 22798, 22821, 22823,22825, 22746, 22748, 22800, 22802, 22804, 22827, 22829, 22831, 22750,22752, 22806, 22808, 22810, 22833, 22835, 22837, 22754, 22756, 22812,23517-23624 22839, 23297-23404 22758, 22947- 22964 3 Stabilized spikeprotein; 408-541 12339, 12340, 12542, 12811, 12812, 13014, S_stab_PP_cav12543 13015 4 Stabilized spike protein; 542-608 12341, 12544 12813,13016 S_stab_PP_prot 5 Stabilized spike protein; S_stab_disul 19-26,609- 12342-12351, 12545- 12814-12823, 13017- 1278, 13521- 12554, 14142,14151 13026, 14160, 14169 13587 6 Spike protein fragment S1 27,1279-1345 152-154, 159-161, 166-168, 173-175, 12352, 12555 12824, 130277 S_woTM comprising a lumazine 58-66, 3624- 12353, 12556 12825, 13028synthase  3690 8 S_stab__PP_woTM comprising a 85-93, 3691- 12354, 1255712826, 13029 lumazine synthase  3757 9 S_stab_PP_cav_woTM comprising a3758-3891 12355, 12356, 12558, 12827, 12828, 13030, lumazine synthase12559 13031 10 S_stab_PP_prot_woTM comprising a 3892-3958 12357, 1256012829, 13032 lumazine synthase 11 S_stab_disul_woTM comprising a 3959-4628, 12358-12367, 12561- 12830-12839, 13033- lumazine synthase13588-13654 12570, 14143, 14152 13042, 14161, 14170 12 S_woTM comprisinga ferritin 67-75, 4629- 12368, 12571 12840, 13043  4695 13S_stab__PP_woTM comprising a ferritin 94-102, 4696- 12369, 12572 12841,13044  4762 14 S_stab_PP_cav_woTM comprising a 4763-4896 12370, 12371,12573, 12842, 12843, 13045, ferritin 12574 13046 15 S_stab_PP_prot_woTMcomprising a 4897-4963 12372, 12575 12844, 13047 ferritin 16S_stab_disul_woTM comprising a  4964-5633, 12373-12382, 12576-12845-12854, 13048- ferritin 13655-13721 12585, 14144, 14153 13057,14162, 14171 17 S_woTM comprising a foldon 76-84, 5634- 12383, 1258612855, 13058  5700 18 S_stab_ PP_woTM comprising a foldon 103-111, 5701-12384, 12587 12856, 13059  5767 19 S_stab_PP_cav_woTM comprising a5768-5901 12385, 12386, 12588, 12857, 12858, 13060, foldon 12589 1306120 S_stab_PP_prot_woTM comprising a 5902-5968 12387, 12590 12859, 13062foldon 21 S_stab_disul_woTM comprising a  5969-6638, 12388-12397, 12591-12860-12869, 13063- foldon 13722-13788 12600, 14145, 14154 13072, 14163,14172 22 S_woTM comprising a WhcAg (VLP) 6639-6705 12398, 12601 12870,13073 23 S_stab_ PP_woTM comprising a 6706-6772 12399, 12602 12871,13074 WhcAg (VLP) 24 S_stab_PP_cav_woTM comprising a 6773-6906 12400,12401, 12603, 12872, 12873, 13075, WhcAg (VLP) 12604 13076 25S_stab_PP_prot_woTM comprising a 6907-6973 12402, 12605 12874, 13077WhcAg (VLP) 26 S_stab_disul_woTM comprising a  6974-7643, 12403-12412,12606- 12875-12884, 13078- WhcAg (VLP) 13789-13855 12615, 14146, 1415513087, 14164, 14173 27 S_woTMflex comprising a lumazine 7644-7710 12413,12616 12885, 13088 synthase 28 S_stab__PP_woTMflex comprising a7711-7777 12414, 12617 12886, 13089 lumazine synthase 29S_stab_PP_cav_woTMflex comprising 7778-7911 12415, 12416, 12618, 12887,12888, 13090, a lumazine synthase 12619 13091 30 S_stab_PP_prot_woTMflexcomprising 7912-7978 12417, 12620 12889, 13092 a lumazine synthase 31S_stab_disul_woTMflex comprising a  7979-8648, 12418-12427, 12621-12890-12899, 13093- lumazine synthase 13856-13922 12630, 14147, 1415613102, 14165, 14174 32 S_woTMflex comprising a ferritin 8649-8715 12428,12631 12900, 13103 33 S_stab_PP_woTMflex comprising a 8716-8782 12429,12632 12901, 13104 ferritin 34 S_stab_PP_cav_woTMflex comprising8783-8916 12430, 12431, 12633, 12902, 12903, 13105, a ferritin 1263413106 35 S_stab_PP_prot_woTMflex comprising 8917-8983 12432, 1263512904, 13107 a ferritin 36 S_stab_disul_woTMflex comprising a 8984-9653, 12433-12442, 12636- 12905-12914, 13108- ferritin 13923-1398912645, 14148, 14157 13117, 14166, 14175 37 S_woTMflex comprising afoldon 9654-9720 12443, 12646 12915, 13118 38 S_stab_ PP_woTMflexcomprising a 9721-9787 12444, 12647 12916, 13119 foldon 39S_stab_PP_cav_woTMflex comprising 9788-9921 12445, 12446, 12648, 12917,12918, 13120, a foldon 12649 13121 40 S_stab_PP_prot_woTMflex comprising9922-9988 12447, 12650 12919, 13122 a foldon 41 S_stab_disul_woTMflexcomprising a  9989-10658, 12448-12457, 12651- 12920-12929, 13123- foldon13990-14056 12660, 14149, 14158 13132, 14167, 14176 42 S_woTMflexcomprising a WhcAg (VLP) 10659-10725 12458, 12661 12930, 13133 43S_stab_ PP_woTMflex comprising a 10726-10792 12459, 12662 12931, 13134WhcAg (VLP) 44 S_stab_PP_cav_woTMflex comprising 10793-10926 12460,12461, 12663, 12932, 12933, 13135, a WhcAg (VLP) 12664 13136 45S_stab_PP_prot_woTMflex comprising 10927-10993 12462, 12665 12934, 13137a WhcAg (VLP) 46 S_stab_disul_woTMflex comprising a  10994-11663,12463-12472, 12666- 12935-12944, 13138- WhcAg (VLP) 14057-14123 12675,14150, 14159 13147, 14168, 14177 47 Stabilized spike protein; 2273222786 22813 S_stab_PP_hex 48 RBD comprising a lumazyne synthase 22735,22736 22789, 22790 22816, 22817 49 RBD comprising a ferritin 22733 2278722814 50 RBD comprising a foldon 22734 22788 22815

Further preferred nucleic acid sequences, preferably mRNA sequences ofthe invention are provided in Table 3b. Therein, each column representsa specific suitable SARS-CoV-2 (nCoV-2019) construct of the invention(compare with Table 1 and Table 3a), wherein column B represents“Full-length spike protein; S”, row 1 of Table 1 and Table 3a and columnC “Stabilized spike protein; S_stab_PP”, compare with row 2 of Table 1and Table 3a.

The SEQ ID NOs of the amino acid sequence of the respective SARS-CoV-2construct are provided in row 1. The corresponding SEQ ID NOs of thecoding sequences encoding the respective SARS-CoV-2 constructs areprovided in in Table 1. Further information is provided under <223>identifier of the respective SEQ ID NOs in the sequence listing.

The corresponding nucleic acid, preferably coding RNA sequences, inparticular mRNA sequences comprising preferred coding sequences areprovided in rows 2-16, wherein each row provides nucleic acid sequenceswith UTR combinations and suitable 3′ ends.

TABLE 3b Nucleic acid, preferably mRNA constructs suitable for acoronavirus vaccine row A B C 1 Protein 1-9, 274-340, 22737, 10-18,341-407, 22738, 22739, 22741, 22740, 22742, 22743, 22745, 22747, 22744,22746, 22748, 22749, 22751, 22750, 22752, 22753, 22755, 22757, 22754,22756, 22758, 22929-22946 22947-22964 2 RNA i-3 162, 169, 12676-12809,163-165, 170-172, (A64- 12945-13012, 12810, 13013, 22819, N5-C30- 22818,22820, 22822, 22821, 22823, 22825, hSL-N5) 22824, 22826, 22827, 22829,22828, 22830, 22832, 22831, 22833, 22835, 22834, 22836, 22837, 22839,22838, 23189-23296 23297-23404 3 RNA a-1 148, 155,12204-12337, 149-151,156-158, (hSL-A100) 12473-12540, 12338, 12541, 22792, 22791, 22793,22795, 22794, 22796, 22798, 22797, 22799, 22800, 22802, 22801, 22803,22805, 22804, 22806, 22808, 22807, 22809, 22810, 22812, 22811,23409-23516 23517-23624 4 RNA a-3 23629-23736 23737-23844 (hSL-A100) 5RNA e-2 23849-23956 23957-24064 (hSL-A100) 6 RNA i-3 24069-24176,24177-24284, (hSL-A100) 24289-24396, 24509- 24397-24504, 2461624617-24724 7 RNA a-1 24729-24836 24837-24944 (A100) 8 RNA a-324949-25056 25057-25164 (A100) 9 RNA e-2 25169-25276 25277-25384 (A100)10 RNA i-3 25389-25496, 25497-25604, (A100) 25609-25716, 25829-25717-25824, 25936 25937-26044 13 RNA x-1 26049-26156 26157-26264 (A100)14 RNA x-2 26269-26376 26377-26484 (A100) 15 RNA x-1 26489-2659626597-26704 (A100-N5) 16 RNA x-2 26709-26816 26817-26924 (A30-N10-A70)

In preferred embodiments, the nucleic acid, preferably the RNA,comprises or consists of a nucleic acid sequence which is identical orat least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence selectedfrom the group consisting of SEQ ID NOs: 148-175, 12204-13147,14142-14177, 22786-22839, 23189-23404, 23409-23624, 23629-23844,23849-24064, 24069-24284, 24289-24504, 24509-24724, 24729-24944,24949-25164, 25169-25384, 25389-25604, 25609-25824, 25829-26044,26049-26264, 26269-26484, 26489-26704, 26709-26937 or a fragment orvariant of any of these sequences. Further information regardingrespective nucleic acid sequences is provided under <223> identifier ofthe respective SEQ ID NO in the sequence listing, and in Table 3a (seein particular Column C and D) and Table 3b (see in particular rows2-16).

In particularly preferred embodiments, the nucleic acid, preferably theRNA, comprises or consists of a nucleic acid sequence which is identicalor at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence selectedfrom the group consisting of SEQ ID NOs: 162-175, 12676-13147,14160-14177, 22786-22839, 23189-23404, or a fragment or variant of anyof these sequences. Further information regarding respective nucleicacid sequences is provided under <223> identifier of the respective SEQID NO in the sequence listing, and in Table 3a (see in particular ColumnD), Table 3b (row 2).

In particularly preferred embodiments, the nucleic acid, preferably theRNA, comprises or consists of a nucleic acid sequence which is identicalor at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence selectedfrom the group consisting of SEQ ID NOs: 148-161, 12204-12675,14142-14159, 22786-22812, 23409-23624, 24729-24944, or a fragment orvariant of any of these sequences. Further information regardingrespective nucleic acid sequences is provided under <223> identifier ofthe respective SEQ ID NO in the sequence listing, and in Table 3a (seein particular Column C) and Table 3b (see rows 3, 7).

In particularly preferred embodiments, the nucleic acid, preferably theRNA, comprises or consists of a nucleic acid sequence which is identicalor at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence selectedfrom the group consisting of SEQ ID NOs: 149-154, 156-161, 163-168,170-175, 12338, 12352, 12541, 12555, 12810, 12824, 13013, 13027, 22786,22792, 22794, 22796, 22798, 22800, 22802, 22804, 22806, 22808, 22810,22812, 22813, 22819, 22821, 22823, 22825, 22827, 22829, 22831, 22833,22835, 22837, 22839, 23517-23624, 23297-23404, 24837-24944 or a fragmentor variant of any of these sequences. Further information regardingrespective nucleic acid sequences is provided under <223> identifier ofthe respective SEQ ID NO in the sequence listing and in Table 3a (seeColumn C and D, rows 2 and 6) and Table 3b (see Column C).

In even more preferred embodiments, the nucleic acid, preferably theRNA, comprises or consists of a nucleic acid sequence which is identicalor at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence selectedfrom the group consisting of SEQ ID NOs: 149, 156, 12338, 150, 157, 151,158, 12541, 163, 170, 12810, 164, 171, 165, 172, 13013, 12342-12351,12545-12554, 12814-12823, 13017-13026, 14133 or a fragment or variant ofany of these sequences. Further information regarding respective nucleicacid sequences is provided under <223> identifier of the respective SEQID NO in the sequence listing and in Table 3a and 3b.

In even more preferred embodiments, the nucleic acid, preferably theRNA, comprises or consists of a nucleic acid sequence selected from thegroup consisting of SEQ ID NOs: 149, 150, 151, 163, 164, 165 or afragment or variant of any of these sequences. Further informationregarding respective nucleic acid sequences is provided under <223>identifier of the respective SEQ ID NO in the sequence listing and inTable 3 (see Column C and D, row 2).

In a particularly preferred embodiment, the nucleic acid, preferably theRNA, comprises or consists of a nucleic acid sequence which is identicalor at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence of SEQID NO: 163.

In a further particularly preferred embodiment, the nucleic acid,preferably the RNA, comprises or consists of a nucleic acid sequencewhich is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleicacid sequence of SEQ ID NO: 149.

In a further particularly preferred embodiment, the nucleic acid,preferably the RNA, comprises or consists of a nucleic acid sequencewhich is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleicacid sequence of SEQ ID NO: 24837.

In a further preferred embodiment, the nucleic acid, preferably the RNA,comprises or consists of a nucleic acid sequence which is identical orat least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence of SEQID NO: 23311, 23531, 24851.

In a further preferred embodiment, the nucleic acid, preferably the RNA,comprises or consists of a nucleic acid sequence which is identical orat least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence of SEQID NO: 23310, 23530, 24850.

In a further preferred embodiment, the nucleic acid, preferably the RNA,comprises or consists of a nucleic acid sequence which is identical orat least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence of SEQID NO: 23313, 23533, 24853, 23314, 23534, 24854.

In a further embodiment, the nucleic acid, preferably the RNA, comprisesor consists of a nucleic acid sequence which is identical or at least70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% identical to a nucleic acid sequence of SEQ ID NO:26633.

In a further embodiment, the nucleic acid, preferably the RNA, comprisesor consists of a nucleic acid sequence which is identical or at least70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% identical to a nucleic acid sequence of SEQ ID NO:26907.

In further preferred embodiments, the nucleic acid, preferably the RNA,comprises or consists of a nucleic acid sequence which is identical orat least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence selectedfrom the group consisting of SEQ ID NOs: 148-175, 12204-13147,14142-14177, 22786-22839, 23189-23404, 23409-23624, 23629-23844,23849-24064, 24069-24284, 24289-24504, 24509-24724, 24729-24944,24949-25164, 25169-25384, 25389-25604, 25609-25824, 25829-26044,26049-26264, 26269-26484, 26489-26704, 26709-26937, wherein said RNAsequences comprise a cap1 structure as defined herein. Furtherinformation regarding respective nucleic acid sequences is providedunder <223> identifier of the respective SEQ ID NO in the sequencelisting and in Table 3a and 3b.

In further embodiments, the nucleic acid, preferably the RNA, comprisesor consists of a nucleic acid sequence which is identical or at least70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% identical to a nucleic acid sequence selected from thegroup consisting of SEQ ID NOs: 148-175, 12204-13147, 14142-14177,22786-22839, 23189-23404, 23409-23624, 23629-23844, 23849-24064,24069-24284, 24289-24504, 24509-24724, 24729-24944, 24949-25164,25169-25384, 25389-25604, 25609-25824, 25829-26044, 26049-26264,26269-26484, 26489-26704, 26709-26937, wherein at least one, preferablyall uracil nucleotides in said RNA sequences are replaced bypseudouridine (ψ) nucleotides and/or N1-methylpseudouridine (m1ψ)nucleotides. Further information regarding respective nucleic acidsequences is provided under <223> identifier of the respective SEQ ID NOin the sequence listing and in Table 3a and 3b.

In further embodiments, the nucleic acid, preferably the RNA, comprisesor consists of a nucleic acid sequence which is identical or at least70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% identical to a nucleic acid sequence selected from thegroup consisting of SEQ ID NOs: 148-175, 12204-13147, 14142-14177,22786-22839, 23189-23404, 23409-23624, 23629-23844, 23849-24064,24069-24284, 24289-24504, 24509-24724, 24729-24944, 24949-25164,25169-25384, 25389-25604, 25609-25824, 25829-26044, 26049-26264,26269-26484, 26489-26704, 26709-26937, wherein said RNA sequencescomprise a cap1 structure as defined herein, and, wherein at least one,preferably all uracil nucleotides in said RNA sequences are replaced bypseudouridine (ψ) nucleotides and/or N1-methylpseudouridine (m1ψ)nucleotides. Further information regarding respective nucleic acidsequences is provided under <223> identifier of the respective SEQ ID NOin the sequence listing and in Table 3a and 3b.

As outlined throughout the specification, additional informationregarding suitable amino acid sequences or nucleic acid sequences(coding sequences, DNA sequences, RNA sequences) may also be derivedfrom the sequence listing, in particular from the details providedtherein under identifier <223> as explained in the following.

In particular embodiments, the nucleic acid of the invention is an RNA,wherein the RNA may be prepared using any method known in the art,including chemical synthesis such as e.g. solid phase RNA synthesis, aswell as in vitro methods, such as RNA in vitro transcription reactions.Accordingly, in a preferred embodiment, the RNA is obtained by RNA invitro transcription.

Accordingly, in preferred embodiments, the nucleic acid of the inventionis preferably an in vitro transcribed RNA.

The terms “RNA in vitro transcription” or “in vitro transcription”relate to a process wherein RNA is synthesized in a cell-free system (invitro). RNA may be obtained by DNA-dependent in vitro transcription ofan appropriate DNA template, which according to the present invention isa linearized plasmid DNA template or a PCR-amplified DNA template. Thepromoter for controlling RNA in vitro transcription can be any promoterfor any DNA-dependent RNA polymerase. Particular examples ofDNA-dependent RNA polymerases are the T7, T3, SP6, or Syn5 RNApolymerases. In a preferred embodiment of the present invention the DNAtemplate is linearized with a suitable restriction enzyme, before it issubjected to RNA in vitro transcription.

Reagents used in RNA in vitro transcription typically include: a DNAtemplate (linearized plasmid DNA or PCR product) with a promotersequence that has a high binding affinity for its respective RNApolymerase such as bacteriophage-encoded RNA polymerases (T7, T3, SP6,or Syn5); ribonucleotide triphosphates (NTPs) for the four bases(adenine, cytosine, guanine and uracil); optionally, a cap analogue asdefined herein; optionally, further modified nucleotides as definedherein; a DNA-dependent RNA polymerase capable of binding to thepromoter sequence within the DNA template (e.g. T7, T3, SP6, or Syn5 RNApolymerase); optionally, a ribonuclease (RNase) inhibitor to inactivateany potentially contaminating RNase; optionally, a pyrophosphatase todegrade pyrophosphate, which may inhibit RNA in vitro transcription;MgCl2, which supplies Mg2+ ions as a co-factor for the polymerase; abuffer (TRIS or HEPES) to maintain a suitable pH value, which can alsocontain antioxidants (e.g. DTT), and/or polyamines such as spermidine atoptimal concentrations, e.g. a buffer system comprising TRIS-Citrate asdisclosed in WO2017/109161.

In preferred embodiments, the cap1 structure of the RNA of the inventionis formed using co-transcriptional capping using tri-nucleotide capanalogues m7G(5′)ppp(5′)(2′OMeA)pG or m7G(5′)ppp(5′)(2′OMeG)pG. Apreferred cap1 analogue that may suitably be used in manufacturing thecoding RNA of the invention is m7G(5′)ppp(5′)(2′OMeA)pG.

In a particularly preferred embodiment, the cap1 structure of the RNA ofthe invention is formed using co-transcriptional capping usingtri-nucleotide cap analogue 3′OMe-m7G(5′)ppp(5′)(2′OMeA)pG.

In other embodiments, a capO structure of the RNA of the invention isformed using co-transcriptional capping using cap analogue3′OMe-m7G(5′)ppp(5′)G.

In additional embodiments, the nucleotide mixture used in RNA in vitrotranscription may additionally comprise modified nucleotides as definedherein. In that context, preferred modified nucleotides may be selectedfrom pseudouridine (ψ), N1-methylpseudouridine (m1ψ), 5-methylcytosine,and 5-methoxyuridine. In particular embodiments, uracil nucleotides inthe nucleotide mixture are replaced (either partially or completely) bypseudouridine (ψ) and/or N1-methylpseudouridine (m1ψ) to obtain amodified RNA.

In preferred embodiments, the nucleotide mixture used in RNA in vitrotranscription does not comprise modified nucleotides as defined herein.In preferred embodiments, the nucleotide mixture used in RNA in vitrotranscription does only comprise G, C, A and U nucleotides, and,optionally, a cap analog as defined herein.

In preferred embodiments, the nucleotide mixture (i.e. the fraction ofeach nucleotide in the mixture) used for RNA in vitro transcriptionreactions may be optimized for the given RNA sequence, preferably asdescribed WO2015/188933. In this context the in vitro transcription hasbeen performed in the presence of a sequence optimized nucleotidemixture and optionally a cap analog, preferably wherein the sequenceoptimized nucleotide mixture does not comprise chemically modifiednucleotides.

In this context a sequence-optimized nucleoside triphosphate (NTP) mixis a mixture of nucleoside triphosphates (NTPs) for use in an in vitrotranscription reaction of an RNA molecule of a given sequence comprisingthe four nucleoside triphosphates (NTPs) GTP, ATP, CTP and UTP, whereinthe fraction of each of the four nucleoside triphosphates (NTPs) in thesequence-optimized nucleoside triphosphate (NTP) mix corresponds to thefraction of the respective nucleotide in said RNA molecule. If aribonucleotide is not present in the RNA molecule, the correspondingnucleoside triphosphate is also not present in the sequence-optimizednucleoside triphosphate (NTP) mix.

In embodiments where more than one different RNA as defined herein haveto be produced, e.g. where 2, 3, 4, 5, 6, 7, 8, 9, 10 or even moredifferent RNAs have to be produced (see second aspect), procedures asdescribed in WO2017/109134 may suitably be used.

In the context of nucleic acid-based vaccine production, it may berequired to provide GMP-grade nucleic acids, e.g. a GMP grade RNA orDNA. GMP-grade RNA or DNA may be produced using a manufacturing processapproved by regulatory authorities. Accordingly, in a particularlypreferred embodiment, RNA production is performed under current goodmanufacturing practice (GMP), implementing various quality control stepson DNA and RNA level, preferably according to WO2016/180430. Inpreferred embodiments, the RNA of the invention is a GMP-grade RNA,particularly a GMP-grade mRNA. Accordingly, an RNA for a vaccine ispreferably a GMP grade RNA.

The obtained RNA products are preferably purified using PureMessenger®(CureVac, Tubingen, Germany; RP-HPLC according to WO2008/077592) and/ortangential flow filtration (as described in WO2016/193206) and/or oligod(T) purification (see WO2016/180430).

Preferably, the RNA according to the invention is purified usingRP-HPLC, preferably using Reversed-Phase High pressure liquidchromatography (RP-HPLC) with a macroporous styrene/divinylbenzenecolumn (e.g. particle size 30 μm, pore size 4000 Å and additionallyusing a filter cassette with a cellulose based membrane with a molecularweight cutoff of about 100 kDa.

In this context it is particularly preferred that the purified RNA hasbeen purified by RP-HPLC and/or TFF which results in about 5%, 10%, or20% less double stranded RNA side products as in RNA that has not beenpurified with RP-HPLC and/or TFF.

Alternatively, the purified RNA that has been purified by RP-HPLC and/orTFF comprises about 5%, 10%, or 20% less double stranded RNA sideproducts as an RNA that has been purified with Oligo dT purification,precipitation, filtration and/or anion exchange chromatography.

In a further preferred embodiment, the nucleic acid, preferably the RNA,is lyophilized (e.g. according to WO2016/165831 or WO2011/069586) toyield a temperature stable dried nucleic acid (powder) as defined herein(e.g. RNA or DNA). The nucleic acid of the invention, particularly theRNA may also be dried using spray-drying or spray-freeze drying (e.g.according to WO2016/184575 or WO2016/184576) to yield a temperaturestable RNA (powder) as defined herein. Accordingly, in the context ofmanufacturing and purifying nucleic acid, in particular RNA, thedisclosures of WO2017/109161, WO2015/188933, WO2016/180430,WO2008/077592, WO2016/193206, WO2016/165831, WO2011/069586,WO2016/184575, and WO2016/184576 are incorporated herewith by reference.

Accordingly, in preferred embodiments, the nucleic acid is a driednucleic acid, particularly a dried RNA.

The term “dried RNA” as used herein has to be understood as RNA that hasbeen lyophilized, or spray-dried, or spray-freeze dried as defined aboveto obtain a temperature stable dried RNA (powder).

In preferred embodiments, the nucleic acid of the invention is apurified nucleic acid, particularly a purified RNA.

The term “purified nucleic acid” as used herein has to be understood asnucleic acid which has a higher purity after certain purification stepsthan the starting material. Typical impurities that are essentially notpresent in purified nucleic acid comprise peptides or proteins,spermidine, BSA, abortive nucleic acid sequences, nucleic acidfragments, free nucleotides, bacterial impurities, or impurities derivedfrom purification procedures. Accordingly, it is desirable in thisregard for the “degree of nucleic acid purity” to be as close aspossible to 100%. It is also desirable for the degree of nucleic acidpurity that the amount of full-length nucleic acid is as close aspossible to 100%. Accordingly “purified nucleic acid” as used herein hasa degree of purity of more than 75%, 80%, 85%, very particularly 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and most favorably 99% or more.The degree of purity may for example be determined by an analyticalHPLC, wherein the percentages provided above correspond to the ratiobetween the area of the peak for the target nucleic acid and the totalarea of all peaks representing the by-products. Alternatively, thedegree of purity may for example be determined by an analytical agarosegel electrophoresis or capillary gel electrophoresis.

In preferred embodiments, the nucleic acid of the invention is apurified RNA.

The term “purified RNA” or “purified mRNA” as used herein has to beunderstood as RNA which has a higher purity after certain purificationsteps (e.g. HPLC, TFF, Oligo d(T) purification, precipitation steps)than the starting material (e.g. in vitro transcribed RNA). Typicalimpurities that are essentially not present in purified RNA comprisepeptides or proteins (e.g. enzymes derived from DNA dependent RNA invitro transcription, e.g. RNA polymerases, RNases, pyrophosphatase,restriction endonuclease, DNase), spermidine, BSA, abortive RNAsequences, RNA fragments (short double stranded RNA fragments, abortivesequences etc.), free nucleotides (modified nucleotides, conventionalNTPs, cap analogue), template DNA fragments, buffer components (HEPES,TRIS, MgCl2) etc. Other potential impurities that may be derived frome.g. fermentation procedures comprise bacterial impurities (bioburden,bacterial DNA) or impurities derived from purification procedures(organic solvents etc.). Accordingly, it is desirable in this regard forthe “degree of RNA purity” to be as close as possible to 100%. It isalso desirable for the degree of RNA purity that the amount offull-length RNA transcripts is as close as possible to 100%.Accordingly, “purified RNA” as used herein has a degree of purity ofmore than 75%, 80%, 85%, very particularly 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% and most favorably 99% or more. The degree of purity mayfor example be determined by an analytical HPLC, wherein the percentagesprovided above correspond to the ratio between the area of the peak forthe target RNA and the total area of all peaks representing theby-products. Alternatively, the degree of purity may for example bedetermined by an analytical agarose gel electrophoresis or capillary gelelectrophoresis.

In particularly preferred embodiments where the nucleic acid is an RNA,the RNA has been purified by RP-HPLC and/or TFF to removedouble-stranded RNA, non-capped RNA and/or RNA fragments.

The formation of double stranded RNA as side products during e.g. RNA invitro transcription can lead to an induction of the innate immuneresponse, particularly IFNalpha which is the main factor of inducingfever in vaccinated subjects, which is of course an unwanted sideeffect. Current techniques for immunoblotting of dsRNA (via dot Blot,serological specific electron microscopy (SSEM) or ELISA for example)are used for detecting and sizing dsRNA species from a mixture ofnucleic acids.

Suitably, the RNA of the invention has been purified by RP-HPLC and/orTFF as described herein to reduce the amount of dsRNA.

In preferred embodiments, the RNA of the invention comprises about 5%,10%, or 20% less double stranded RNA side products as an RNA that hasnot been purified with RP-HPLC and/or TFF.

In preferred embodiments, the RP-HPLC and/or TFF purified RNA of theinvention comprises about 5%, 10%, or 20% less double stranded RNA sideproducts as an RNA that has been purified with Oligo dT purification,precipitation, filtration and/or AEX.

It has to be understood that “dried RNA” as defined herein and “purifiedRNA” as defined herein or “GMP-grade RNA” as defined herein may havesuperior stability characteristics (in vitro, in vivo) and improvedefficiency (e.g. better translatability of the mRNA in vivo) and aretherefore particularly suitable for a medical purpose, e.g. a vaccine.

Following co-transcriptional capping as defined herein, and followingpurification as defined herein, the capping degree of the obtained RNAmay be determined using capping assays as described in published PCTapplication WO2015/101416, in particular, as described in claims 27 to46 of published PCT application WO2015/101416 can be used.Alternatively, a capping assays described in PCT/EP2018/08667 may beused.

In embodiments, an automated device for performing RNA in vitrotranscription may be used to produce and purify the nucleic acid of theinvention. Such a device may also be used to produce the composition orthe vaccine (see aspects 2 and 3). Preferably, a device as described inWO2020002598, in particular, a device as described in claims 1 to 59and/or 68 to 76 of WO2020002598 (and FIGS. 1-18) may suitably be used.

The methods described herein may preferably applied to a method ofproducing an RNA composition or vaccine as described in further detailbelow.

Composition, Pharmaceutical Composition:

A second aspect relates to a composition comprising at least one nucleicacid of the first aspect.

Notably, embodiments relating to the composition of the second aspectmay likewise be read on and be understood as suitable embodiments of thevaccine of the fourth aspect. Also, embodiments relating to the vaccineof the fourth aspect may likewise be read on and be understood assuitable embodiments of the composition of the second aspect (comprisingthe nucleic acid of the first aspect). Furthermore, features andembodiments described in the context of the first aspect (the nucleicacid of the invention) have to be read on and have to be understood assuitable embodiments of the composition of the second aspect.

In preferred embodiments, the composition comprises at least one nucleicacid according to the first aspect encoding at least one antigenicpeptide or protein that is or is derived from a SARS-CoV-2 (formerly annCoV-2019) coronavirus, or an immunogenic fragment or immunogenicvariant thereof.

In preferred embodiments, the composition comprises at least one nucleicacid encoding at least one antigenic peptide or protein that is or isderived from a SARS-CoV-2 coronavirus, or an immunogenic fragment orimmunogenic variant thereof according to the first aspect, wherein saidcomposition is to be, preferably, administered intramuscularly orintradermal.

Preferably, intramuscular or intradermal administration of saidcomposition results in expression of the encoded SARS-CoV-2 antigenconstruct in a subject. In embodiments where the nucleic acid is an RNA,administration of said composition results in translation of the RNA andto a production of the encoded SARS-CoV-2 antigen in a subject. Inembodiments where the nucleic acid is a DNA (e.g. plasmid DNA,adenovirus DNA), administration of said composition results intranscription of the DNA into RNA, and to a subsequent translation ofthe RNA into the encoded SARS-CoV-2 coronavirus antigen in a subject.

Preferably, the composition of the second aspect is suitable for avaccine, in particular, suitable for a coronavirus vaccine, preferably aSARS-CoV-2 (nCoV-2019) vaccine.

In the context of the invention, a “composition” refers to any type ofcomposition in which the specified ingredients (e.g. nucleic acidencoding at least one antigenic peptide or protein that is or is derivedfrom a SARS-CoV-2 coronavirus, e.g. an RNA or a DNA, e.g. in associationwith a polymeric carrier or LNP) may be incorporated, optionally alongwith any further constituents, usually with at least onepharmaceutically acceptable carrier or excipient. The composition may bea dry composition such as a powder or granules, or a solid unit such asa lyophilized form. Alternatively, the composition may be in liquidform, and each constituent may be independently incorporated indissolved or dispersed (e.g. suspended or emulsified) form.

In a preferred embodiment of the second aspect, the compositioncomprises at least one nucleic acid (e.g. DNA or RNA) of the firstaspect, preferably an RNA, and optionally, at least one pharmaceuticallyacceptable carrier or excipient.

In embodiments of the second aspect, the composition comprises at leastone nucleic acid of the first aspect, preferably a plasmid DNA,adenovirus DNA, and optionally, at least one pharmaceutically acceptablecarrier or excipient.

In preferred embodiments of the second aspect, the composition comprisesat least one nucleic acid (e.g. DNA or RNA), preferably an RNA, whereinthe nucleic acid comprises or consists of a nucleic acid sequence whichis identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acidsequence selected from the group consisting of SEQ ID NOs: 116-132,134-138, 140-143, 145-175, 11664-11813, 11815, 11817-12050, 12052,12054-13147, 13514, 13515, 13519, 13520, 14124-14177, 22759,22764-22786, 22791-22813, 22818-22839, 22969-23184, 23189-23404,23409-23624, 23629-23844, 23849-24064, 24069-24284, 24289-24504,24509-24724, 24729-24944, 24949-25164, 25169-25384, 25389-25604,25609-25824, 25829-26044, 26049-26264, 26269-26484, 26489-26704,26709-26937 and, optionally, at least one pharmaceutically acceptablecarrier or excipient.

In particularly preferred embodiments of the second aspect, thecomposition comprises at least one nucleic acid (e.g. DNA or RNA),preferably an RNA, wherein the nucleic acid comprises or consists of anucleic acid sequence which is identical or at least 70%, 80%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to a nucleic acid sequence selected from the group consistingof SEQ ID NOs: 148-175, 12204-13147, 14142-14177, 22786-22839,23189-23404, 23409-23624, 23629-23844, 23849-24064, 24069-24284,24289-24504, 24509-24724, 24729-24944, 24949-25164, 25169-25384,25389-25604, 25609-25824, 25829-26044, 26049-26264, 26269-26484,26489-26704, 26709-26937 and, optionally, at least one pharmaceuticallyacceptable carrier or excipient.

Most preferably the composition comprises at least one nucleic acid(e.g. DNA or RNA), preferably an RNA, wherein the nucleic acid comprisesor consists of a nucleic acid sequence which is identical or at least70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% identical to a nucleic acid sequence of SEQ ID NO. 163.

Most preferably the composition comprises at least one nucleic acid(e.g. DNA or RNA), preferably an RNA, wherein the nucleic acid comprisesor consists of a nucleic acid sequence which is identical or at least70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% identical to a nucleic acid sequence of SEQ ID NO. 149.

In further particularly preferred embodiments, the composition comprisesat least one nucleic acid (e.g. DNA or RNA), preferably an RNA, whereinthe nucleic acid comprises or consists of a nucleic acid sequence whichis identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acidsequence of SEQ ID NO. 24837, 23311, 23531, 23310, 23530, 23313, 23533.

In further embodiments, the composition comprises at least one nucleicacid (e.g. DNA or RNA), preferably an RNA, wherein the nucleic acidcomprises or consists of a nucleic acid sequence which is identical orat least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence of SEQID NO. 26633, 26907.

In particularly preferred embodiments of the second aspect, thecomposition comprises at least one nucleic acid (e.g. DNA or RNA),preferably an RNA, wherein the nucleic acid comprises or consists of anucleic acid sequence which is identical or at least 70%, 80%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to a nucleic acid sequence selected from the group consistingof SEQ ID NOs: 149-151, 163-165, 24837, 23311, 23531, 24851, 23310,23530, 23313, 23533 and, optionally, at least one pharmaceuticallyacceptable carrier or excipient.

The term “pharmaceutically acceptable carrier” or “pharmaceuticallyacceptable excipient” as used herein preferably includes the liquid ornon-liquid basis of the composition for administration. If thecomposition is provided in liquid form, the carrier may be water, e.g.pyrogen-free water; isotonic saline or buffered (aqueous) solutions,e.g. phosphate, citrate etc. buffered solutions. Water or preferably abuffer, more preferably an aqueous buffer, may be used, containing asodium salt, preferably at least 50 mM of a sodium salt, a calcium salt,preferably at least 0.01 mM of a calcium salt, and optionally apotassium salt, preferably at least 3 mM of a potassium salt. Accordingto preferred embodiments, the sodium, calcium and, optionally, potassiumsalts may occur in the form of their halogenides, e.g. chlorides,iodides, or bromides, in the form of their hydroxides, carbonates,hydrogen carbonates, or sulfates, etc. Examples of sodium salts includeNaCl, NaI, NaBr, Na₂CO₃, NaHCO₃, Na₂SO₄, examples of the optionalpotassium salts include KCl, KI, KBr, K₂CO₃, KHCO₃, K₂SO₄, and examplesof calcium salts include CaCl₂), CaI₂, CaBr₂, CaCO₃, CaSO₄, Ca(OH)₂.

Furthermore, organic anions of the aforementioned cations may be in thebuffer. Accordingly, in embodiments, the nucleic acid composition maycomprise pharmaceutically acceptable carriers or excipients using one ormore pharmaceutically acceptable carriers or excipients to e.g. increasestability, increase cell transfection, permit the sustained or delayed,increase the translation of encoded coronavirus protein in vivo, and/oralter the release profile of encoded coronavirus protein in vivo. Inaddition to traditional excipients such as any and all solvents,dispersion media, diluents, or other liquid vehicles, dispersion orsuspension aids, surface active agents, isotonic agents, thickening oremulsifying agents, preservatives, excipients of the present inventioncan include, without limitation, lipidoids, liposomes, lipidnanoparticles, polymers, lipoplexes, core-shell nanoparticles, peptides,proteins, cells transfected with polynucleotides, hyaluronidase,nanoparticle mimics and combinations thereof. In embodiments, one ormore compatible solid or liquid fillers or diluents or encapsulatingcompounds may be used as well, which are suitable for administration toa subject. The term “compatible” as used herein means that theconstituents of the composition are capable of being mixed with the atleast one nucleic acid and, optionally, a plurality of nucleic acids ofthe composition, in such a manner that no interaction occurs, whichwould substantially reduce the biological activity or the pharmaceuticaleffectiveness of the composition under typical use conditions (e.g.,intramuscular or intradermal administration). Pharmaceuticallyacceptable carriers or excipients must have sufficiently high purity andsufficiently low toxicity to make them suitable for administration to asubject to be treated. Compounds which may be used as pharmaceuticallyacceptable carriers or excipients may be sugars, such as, for example,lactose, glucose, trehalose, mannose, and sucrose; starches, such as,for example, corn starch or potato starch; dextrose; cellulose and itsderivatives, such as, for example, sodium carboxymethylcellulose,ethylcellulose, cellulose acetate; powdered tragacanth; malt; gelatin;tallow; solid glidants, such as, for example, stearic acid, magnesiumstearate; calcium sulfate; vegetable oils, such as, for example,groundnut oil, cottonseed oil, sesame oil, olive oil, corn oil and oilfrom theobroma; polyols, such as, for example, polypropylene glycol,glycerol, sorbitol, mannitol and polyethylene glycol; alginic acid.

The at least one pharmaceutically acceptable carrier or excipient of thecomposition may preferably be selected to be suitable for intramuscularor intradermal delivery/administration of said composition. Accordingly,the composition is preferably a pharmaceutical composition, suitably acomposition for intramuscular administration.

Subjects to which administration of the compositions, preferably thepharmaceutical composition, is contemplated include, but are not limitedto, humans and/or other primates; mammals, including commerciallyrelevant mammals such as cattle, pigs, horses, sheep, cats, dogs, mice,and/or rats; and/or birds, including commercially relevant birds such aspoultry, chickens, ducks, geese, and/or turkeys.

Pharmaceutical compositions of the present invention may suitably besterile and/or pyrogen-free.

Multivalent Compositions of the Invention:

In embodiments, the composition (e.g. multivalent composition) asdefined herein may comprise a plurality or at least more than one of thenucleic acid species, e.g. RNA species as defined in the context of thefirst aspect of the invention. Preferably, the composition as definedherein may comprise 2, 3, 4, 5, 6, 7, 8, 9, or 10 different nucleicacids each defined in the context of the first aspect.

In embodiments, the composition (e.g. multivalent composition) maycomprise at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or even more differentnucleic acid species as defined in the context of the first aspect, eachencoding at least one antigenic peptide or protein derived from the samecoronavirus, or a fragment or variant thereof. Particularly, said(genetically) same coronavirus expresses (essentially) the samerepertoire of proteins or peptides, wherein all proteins or peptideshave (essentially) the same amino acid sequence. Particularly, said(genetically) same coronavirus expresses essentially the same proteins,peptides or polyproteins, wherein these protein, peptide or polyproteinspreferably do not differ in their amino acid sequence(s).

In embodiments, the composition (e.g. multivalent composition) comprisesat least 2, 3, 4, 5, 6, 7, 8, 9, 10 or even more different nucleic acidspecies as defined in the context of the first aspect, each encoding atleast one peptide or protein derived from a genetically differentcoronavirus (e.g. a different coronavirus isolate), or a fragment orvariant thereof. The terms “different” or “different coronavirus” asused throughout the present specification have to be understood as thedifference between at least two respective coronavirus (e.g. a differentcoronavirus isolates), wherein the difference is manifested on thegenome of the respective different coronavirus. Particularly, said(genetically) different coronavirus may express at least one differentprotein, peptide or polyprotein, wherein the at least one differentprotein, peptide or polyprotein differs in at least one amino acid.

In preferred embodiments the plurality or at least more than one of thenucleic acid sequences of the multivalent composition each encode adifferent spike protein, preferably a prefusion stabilized spikeprotein.

In this context it is particularly preferred that the different spikeproteins or prefusion stabilized spike proteins are derived fromdifferent SARS-CoV-2 virus variants/isolates, wherein it is particularlypreferred that the spike proteins are derived from B.1.1.7, B.1.351,P.1, or CAL.20C.

In this context it is further preferred that the different spikeproteins or prefusion stabilized spike proteins have amino acid changesin the spike protein comprising:

-   (i) delH69, delV70, Y453F, D614G, 1692V and M1229I;-   (ii) delH69, delV70, delY144, N501Y, A570D, D614G, P681H, T716I,    S982A and D1118H;-   (iii) L18F, D80A, D215G, delL242, delA243, delL244, R2461, K417N,    E484K, N501Y, D614G and A701V;-   (iv) L18F, T20N, P26S, D138Y, R190S, K417T, E484K, N501Y, D614G,    H655Y and T10271; and/or-   (v) S13I, W152C, L452R, and D614G.

In embodiments, the composition (e.g. multivalent composition) comprises2, 3, 4 or 5 nucleic acid species (e.g. DNA or RNA), preferably RNAspecies, wherein said nucleic acid species comprise or consist of anucleic acid sequence which is identical or at least 70%, 80%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to a nucleic acid sequence selected from the group consistingof SEQ ID NOs: 116-132, 134-138, 140-143, 145-175, 11664-11813, 11815,11817-12050, 12052, 12054-13147, 13514, 13515, 13519, 13520,14124-14177, 22759, 22764-22786, 22791-22813, 22818-22839, 22969-23184,23189-23404, 23409-23624, 23629-23844, 23849-24064, 24069-24284,24289-24504, 24509-24724, 24729-24944, 24949-25164, 25169-25384,25389-25604, 25609-25824, 25829-26044, 26049-26264, 26269-26484,26489-26704, 26709-26937 and, optionally, at least one pharmaceuticallyacceptable carrier or excipient, wherein each of the 2, 3, 4 or 5nucleic acid species encode a different antigenic peptide or protein ofa SARS-CoV-2 coronavirus.

Accordingly, in embodiments, the composition (e.g. multivalentcomposition) comprises two nucleic acid species (e.g. DNA or RNA),preferably RNA species, wherein the nucleic acid species comprise orconsist of a nucleic acid sequence which is identical or at least 70%,80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identical to a nucleic acid sequence selected from the groupconsisting of SEQ ID NOs: 148-175, 12204-13147, 14142-14177,22786-22839, 23189-23404, 23409-23624, 23629-23844, 23849-24064,24069-24284, 24289-24504, 24509-24724, 24729-24944, 24949-25164,25169-25384, 25389-25604, 25609-25824, 25829-26044, 26049-26264,26269-26484, 26489-26704, 26709-26937 and, optionally, at least onepharmaceutically acceptable carrier or excipient, wherein each of thetwo nucleic acid species encode a different antigenic peptide or proteinof a SARS-CoV-2 coronavirus.

In embodiments, the composition (e.g. multivalent composition) comprisesthree nucleic acid species (e.g. DNA or RNA), preferably RNA species,wherein the nucleic acid comprises or consists of a nucleic acidsequence which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to anucleic acid sequence selected from the group consisting of SEQ ID NOs:148-175, 12204-13147, 14142-14177, 22786-22839, 23189-23404,23409-23624, 23629-23844, 23849-24064, 24069-24284, 24289-24504,24509-24724, 24729-24944, 24949-25164, 25169-25384, 25389-25604,25609-25824, 25829-26044, 26049-26264, 26269-26484, 26489-26704,26709-26937 and, optionally, at least one pharmaceutically acceptablecarrier or excipient, wherein each of the 2, 3, 4 or 5 nucleic acidspecies encode a different antigenic peptide or protein of a SARS-CoV-2coronavirus.

In the following, particularly preferred embodiments of a multivalentcomposition are provided.

Preferably, the at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or even moredifferent nucleic acid species of the multivalent composition eachencode a different prefusion stabilized spike protein (as defined in thefirst aspect). Preferably, stabilization of the prefusion conformationis obtained by introducing two consecutive proline substitutions atresidues K986 and V987 in the spike protein (Amino acid positionsaccording to reference SEQ ID NO: 1). Accordingly, in preferredembodiments, the at least 2, 3, 4, 5, 6, 7, 8, 9, 10 pre-fusionstabilized spike proteins (S_stab) each comprises at least one prefusionstabilizing mutation, wherein the at least one prefusion stabilizingmutation comprises the following amino acid substitutions: K986P andV987P (amino acid positions according to reference SEQ ID NO: 1).

Accordingly, the at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or even moredifferent nucleic acid species of the multivalent composition eachencode a different prefusion stabilized spike protein, wherein the atleast 2, 3, 4, 5, 6, 7, 8, 9, 10 or even more stabilized spike proteinsare selected from amino acid sequences being identical or at least 70%,80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identical to any one of SEQ ID NOs: 10-26, 341-407,609-1278, 13521-13587, 22738, 22740, 22742, 22744, 22746, 22748, 22750,22752, 22754, 22756, 22758, 22947-22964 or an immunogenic fragment orimmunogenic variant of any of these.

In preferred embodiments, the multivalent composition comprises onenucleic acid species comprising a coding sequence encoding an amino acidsequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any oneof SEQ ID NOs: 10, wherein the multivalent composition additionallycomprises at least 2, 3, 4 further RNA species selected from

-   i) one nucleic acid species comprises a coding sequence encoding an    amino acid sequence being identical or at least 70%, 80%, 85%, 86%,    87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%    identical to any one of SEQ ID NOs: 22961; and/or-   ii) one nucleic acid species comprises a coding sequence encoding an    amino acid sequence being identical or at least 70%, 80%, 85%, 86%,    87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%    identical to any one of SEQ ID NOs: 22960; and/or-   iii) one nucleic acid species comprises a coding sequence encoding    an amino acid sequence being identical or at least 70%, 80%, 85%,    86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or    99% identical to any one of SEQ ID NOs: 22963; and/or-   iv) one nucleic acid species comprises a coding sequence encoding an    amino acid sequence being identical or at least 70%, 80%, 85%, 86%,    87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%    identical to any one of SEQ ID NOs: 22941; and/or-   v) one nucleic acid species comprises a coding sequence encoding an    amino acid sequence being identical or at least 70%, 80%, 85%, 86%,    87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%    identical to any one of SEQ ID NOs: 22964.

In preferred embodiments, the multivalent composition comprises at leasttwo nucleic acid species comprising a coding sequence encoding an aminoacid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical toany one of SEQ ID NOs: 10, 22961; 22960, 22963, 22941, 22964.

Preferably, the at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or even moredifferent nucleic acid species of the multivalent composition comprisenucleic acid coding sequences each encoding a different prefusionstabilized spike protein, wherein the at least 2, 3, 4, 5, 6, 7, 8, 9,10 or even more nucleic acid coding sequences are selected from nucleicacid sequences being identical or at least 70%, 80%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical toany one of SEQ ID NOs: 136-138, 140-143, 145-175, 11731-11813, 11815,11817-12050, 12052, 12054-12203, 13514, 13515, 13519, 13520,14124-14141, 22759, 22764-22785, 22969-23184 or fragments or variants ofany of these.

In preferred embodiments, the multivalent composition comprises onenucleic acid species comprising a coding sequence being identical or atleast 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 137, whereinthe multivalent composition additionally comprises at least 2, 3, 4further RNA species selected from

-   i) one nucleic acid species comprises a coding sequence being    identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,    92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of    SEQ ID NOs: 23091; and/or-   ii) one nucleic acid species comprises a coding sequence being    identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,    92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of    SEQ ID NOs: 23090; and/or-   iii) one nucleic acid species comprises a coding sequence being    identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,    92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of    SEQ ID NOs: 23093; and/or-   iv) one nucleic acid species comprises a coding sequence being    identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,    92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of    SEQ ID NOs: 22999; and/or-   v) one nucleic acid species comprises a coding sequence being    identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,    92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of    SEQ ID NOs: 23094.

Preferably, the at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or even moredifferent nucleic acid species of the multivalent composition comprisenucleic acid coding sequences each encoding a different prefusionstabilized spike protein, wherein the at least 2, 3, 4, 5, 6, 7, 8, 9,10 or even more nucleic acid coding sequences are selected from RNAsequences being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any oneof SEQ ID NOs: 149-151, 163-165, 12338, 12541, 12810-12813, 12901,12931, 13013, 22792, 22794, 22796, 22798, 22802, 22804, 22806, 22810,22813, 22819, 22821, 22823, 22825, 22827, 22829, 22831, 22833, 22835,22837, 22839, 23297-23314, 23369, 23517-23520, 23523-23525, 23527,23529, 23530, 23589, 23737, 23957, 24397, 24837, 25057, 25277, 25717,26925-26937 or fragments or variants of any of these.

In preferred embodiments, the multivalent composition comprises one RNAspecies comprising or consisting of an RNA sequence being identical orat least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 163,wherein the multivalent composition additionally comprises at least 2,3, 4 further RNA species selected from

-   i) one RNA species comprising or consisting of an RNA sequence being    identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,    92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of    SEQ ID NOs: 23311; and/or-   ii) one RNA species comprises a coding sequence being identical or    at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,    95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs:    23310; and/or-   iii) one RNA species comprises a coding sequence being identical or    at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,    95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs:    23313; and/or-   iv) one RNA species comprises a coding sequence being identical or    at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,    95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: SEQ    ID NO: 23219; and/or-   v) one RNA species comprises a coding sequence being identical or at    least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,    95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs:    23314;

wherein, preferably, each of the mRNA species comprise a Cap1 structure,and, optionally, each of the mRNA species do not comprise modifiednucleotides.

In preferred embodiments, the multivalent composition comprises one RNAspecies comprising or consisting of an RNA sequence being identical orat least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 149 or24837, wherein the multivalent composition additionally comprises atleast 2, 3, 4 further RNA species selected from

-   i) one RNA species comprising or consisting of an RNA sequence being    identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,    92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of    SEQ ID NOs: 23531 or 24851; and/or-   ii) one RNA species comprises a coding sequence being identical or    at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,    95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 23530    or 24850; and/or-   iii) one RNA species comprises a coding sequence being identical or    at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,    95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 23533    or 24853; and/or-   iv) one RNA species comprises a coding sequence being identical or    at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,    95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 23439    or 24759; and/or-   v) one RNA species comprises a coding sequence being identical or at    least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,    95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 23534    or 24854;

wherein, preferably, each of the mRNA species comprise a Cap1 structure,and, optionally, each of the mRNA species do not comprise modifiednucleotides.

In further preferred embodiments, the multivalent composition comprisesat least two RNA species being identical or at least 70%, 80%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to any one of SEQ ID NOs: 149 or 24837, 23531 or 24851, 23530or 24850, 23533 or 24853, 23439 or 24759 or 23534 or 24854.

In embodiments, the nucleic acid (e.g. DNA or RNA), preferably RNAspecies of the multivalent composition may be formulated separately(formulation as specified below). In preferred embodiments, the nucleicacid (e.g. DNA or RNA), preferably RNA species of the multivalentcomposition may be co-formulated separately (formulation as specifiedbelow).

Complexation:

In a preferred embodiment of the second aspect, the at least one nucleicacid (e.g. DNA or RNA), preferably the at least one RNA, is complexed orassociated with further compound to obtain a formulated composition. Aformulation in that context may have the function of a transfectionagent. A formulation in that context may also have the function ofprotecting the nucleic acid from degradation.

In a preferred embodiment of the second aspect, the at least one nucleicacid (e.g. DNA or RNA), preferably the at least one RNA, and optionallythe at least one further nucleic acid, is complexed or associated with,or at least partially complexed or partially associated with one or morecationic or polycationic compound, preferably cationic or polycationicpolymer, cationic or polycationic polysaccharide, cationic orpolycationic lipid, cationic or polycationic protein, cationic orpolycationic peptide, or any combinations thereof.

The term “cationic or polycationic compound” as used herein will berecognized and understood by the person of ordinary skill in the art,and are for example intended to refer to a charged molecule, which ispositively charged at a pH value ranging from about 1 to 9, at a pHvalue ranging from about 3 to 8, at a pH value ranging from about 4 to8, at a pH value ranging from about 5 to 8, more preferably at a pHvalue ranging from about 6 to 8, even more preferably at a pH valueranging from about 7 to 8, most preferably at a physiological pH, e.g.ranging from about 7.2 to about 7.5. Accordingly, a cationic component,e.g. a cationic peptide, cationic protein, cationic polymer, cationicpolysaccharide, cationic lipid may be any positively charged compound orpolymer which is positively charged under physiological conditions. A“cationic or polycationic peptide or protein” may contain at least onepositively charged amino acid, or more than one positively charged aminoacid, e.g. selected from Arg, His, Lys or Orn. Accordingly,“polycationic” components are also within the scope exhibiting more thanone positive charge under the given conditions.

Cationic or polycationic compounds, being particularly preferred in thiscontext may be selected from the following list of cationic orpolycationic peptides or proteins of fragments thereof: protamine,nucleoline, spermine or spermidine, or other cationic peptides orproteins, such as poly-L-lysine (PLL), poly-arginine, basicpolypeptides, cell penetrating peptides (CPPs), including HIV-bindingpeptides, HIV-1 Tat (HIV), Tat-derived peptides, Penetratin, VP22derived or analog peptides, HSV VP22 (Herpes simplex), MAP, KALA orprotein transduction domains (PTDs), PpT620, prolin-rich peptides,arginine-rich peptides, lysine-rich peptides, MPG-peptide(s), Pep-1,L-oligomers, Calcitonin peptide(s), Antennapedia-derived peptides,pAntp, p151, FGF, Lactoferrin, Transportan, Buforin-2, Bac715-24, SynB,SynB(1), pVEC, hCT-derived peptides, SAP, or histones. More preferably,the nucleic acid (e.g. DNA or RNA), e.g. the coding RNA, preferably themRNA, is complexed with one or more polycations, preferably withprotamine or oligofectamine, most preferably with protamine.

In preferred embodiment, the at least one nucleic acid (e.g. DNA orRNA), preferably the at least one RNA is complexed with protamine.

Further preferred cationic or polycationic compounds, which can be usedas transfection or complexation agent may include cationicpolysaccharides, for example chitosan, polybrene etc.; cationic lipids,e.g. DOTMA, DMRIE, di-C14-amidine, DOTIM, SAINT, DC-Chol, BGTC, CTAP,DOPC, DODAP, DOPE: Dioleyl phosphatidylethanol-amine, DOSPA, DODAB,DOIC, DMEPC, DOGS, DIMRI, DOTAP, DC-6-14, CLIP1, CLIP6, CLIP9,oligofectamine; or cationic or polycationic polymers, e.g. modifiedpolyaminoacids, such as beta-aminoacid-polymers or reversed polyamides,etc., modified polyethylenes, such as PVP etc., modified acrylates, suchas pDMAEMA etc., modified amidoamines such as pAMAM etc., modifiedpolybetaaminoester (PBAE), such as diamine end modified 1,4 butanedioldiacrylate-co-5-amino-1-pentanol polymers, etc., dendrimers, such aspolypropylamine dendrimers or pAMAM based dendrimers, etc.,polyimine(s), such as PEI, poly(propyleneimine), etc., polyallylamine,sugar backbone based polymers, such as cyclodextrin based polymers,dextran based polymers, etc., silan backbone based polymers, such asPMOXA-PDMS copolymers, etc., blockpolymers consisting of a combinationof one or more cationic blocks (e.g. selected from a cationic polymer asmentioned above) and of one or more hydrophilic or hydrophobic blocks(e.g. polyethyleneglycole); etc.

In this context it is particularly preferred that the at least onenucleic acid (e.g. DNA or RNA), preferably the at least one RNA iscomplexed or at least partially complexed with a cationic orpolycationic compound and/or a polymeric carrier, preferably cationicproteins or peptides. In this context, the disclosure of WO2010/037539and WO2012/113513 is incorporated herewith by reference. Partially meansthat only a part of the nucleic acid is complexed with a cationiccompound and that the rest of the nucleic acid is in uncomplexed form(“free”).

In embodiments, the composition comprises at least one nucleic acid(e.g. DNA or RNA), preferably at least one RNA, complexed with one ormore cationic or polycationic compounds, preferably protamine, and atleast one free (non-complexed) nucleic acid.

In this context it is particularly preferred that the at least onenucleic acid (e.g. DNA or RNA), preferably the at least one RNA iscomplexed, or at least partially complexed with protamine. Preferably,the molar ratio of the nucleic acid, particularly the RNA of theprotamine-complexed RNA to the free RNA may be selected from a molarratio of about 0.001:1 to about 1:0.001, including a ratio of about 1:1.Suitably, the complexed RNA is complexed with protamine by addition ofprotamine-trehalose solution to the RNA sample at a RNA:protamine weightto weight ratio (w/w) of 2:1.

Further preferred cationic or polycationic proteins or peptides that maybe used for complexation can be derived from formula(Arg)I;(Lys)m;(His)n;(Orn)o;(Xaa)x of the patent applicationWO2009/030481 or WO2011/026641, the disclosure of WO2009/030481 orWO2011/026641 relating thereto incorporated herewith by reference.

In preferred embodiments, the at least one nucleic acid (e.g. DNA orRNA), preferably the at least one RNA is complexed, or at leastpartially complexed, with at least one cationic or polycationic proteinsor peptides preferably selected from SEQ ID NOs: 269 to 273, or anycombinations thereof.

According to various embodiments, the composition of the presentinvention comprises at least one nucleic acid (e.g. DNA or RNA),preferably at least one RNA as defined in the context of the firstaspect, and a polymeric carrier.

The term “polymeric carrier” as used herein will be recognized andunderstood by the person of ordinary skill in the art, and are e.g.intended to refer to a compound that facilitates transport and/orcomplexation of another compound (e.g. cargo nucleic acid). A polymericcarrier is typically a carrier that is formed of a polymer. A polymericcarrier may be associated to its cargo (e.g. DNA, or RNA) by covalent ornon-covalent interaction. A polymer may be based on different subunits,such as a copolymer.

Suitable polymeric carriers in that context may include, for example,polyacrylates, polyalkycyanoacrylates, polylactide,polylactide-polyglycolide copolymers, polycaprolactones, dextran,albumin, gelatin, alginate, collagen, chitosan, cyclodextrins,protamine, PEGylated protamine, PEGylated PLL and polyethylenimine(PEI), dithiobis(succinimidylpropionate) (DSP),Dimethyl-3,3′-dithiobispropionimidate (DTBP), poly(ethylene imine)biscarbamate (PEIC), poly(L-lysine) (PLL), histidine modified PLL,poly(N-vinylpyrrolidone) (PVP), poly(propylenimine (PPI),poly(amidoamine) (PAMAM), poly(amido ethylenimine) (SS-PAEI),triehtylenetetramine (TETA), poly((3-aminoester),poly(4-hydroxy-L-proine ester) (PHP), poly(allylamine),poly(a[4-aminobutyl]-L-glycolic acid (PAGA), Poly(D,L-lactic-co-glycolidacid (PLGA), Poly(N-ethyl-4-vinylpyridinium bromide), poly(phosphazene)s(PPZ), poly(phosphoester)s (PPE), poly(phosphoramidate)s (PPA),poly(N-2-hydroxypropylmethacrylamide) (pHPMA),poly(2-(dimethylamino)ethyl methacrylate) (pDMAEMA), poly(2-aminoethylpropylene phosphate) PPE_EA), galactosylated chitosan, N-dodecylatedchitosan, histone, collagen and dextran-spermine. In one embodiment, thepolymer may be an inert polymer such as, but not limited to, PEG. In oneembodiment, the polymer may be a cationic polymer such as, but notlimited to, PEI, PLL, TETA, poly(allylamine),Poly(N-ethyl-4-vinylpyridinium bromide), pHPMA and pDMAEMA. In oneembodiment, the polymer may be a biodegradable PEI such as, but notlimited to, DSP, DTBP and PEIC. In one embodiment, the polymer may bebiodegradable such as, but not limited to, histine modified PLL,SS-PAEI, poly((3-aminoester), PHP, PAGA, PLGA, PPZ, PPE, PPA and PPE-EA.

A suitable polymeric carrier may be a polymeric carrier formed bydisulfide-crosslinked cationic compounds. The disulfide-crosslinkedcationic compounds may be the same or different from each other. Thepolymeric carrier can also contain further components. The polymericcarrier used according to the present invention may comprise mixtures ofcationic peptides, proteins or polymers and optionally furthercomponents as defined herein, which are crosslinked by disulfide bonds(via —SH groups).

In this context, polymeric carriers according to formula{(Arg)I;(Lys)m;(His)n;(Orn)o;(Xaa)x(Cys)y} and formula Cys,{(Arg)I;(Lys)m;(His)n;(Orn)o;(Xaa)x}Cys₂ of the patent applicationWO2012/013326 are preferred, the disclosure of WO2012/013326 relatingthereto incorporated herewith by reference.

In embodiments, the polymeric carrier used to complex the at least onenucleic acid (e.g. DNA or RNA), preferably the at least one RNA may bederived from a polymeric carrier molecule according formula(L-P′—S—[S—P²—S]_(n)—S—P³-L) of the patent application WO2011/026641,the disclosure of WO2011/026641 relating thereto incorporated herewithby reference.

In embodiments, the polymeric carrier compound is formed by, orcomprises or consists of the peptide elements CysArg12Cys (SEQ ID NO:269) or CysArg12 (SEQ ID NO: 270) or TrpArg12Cys (SEQ ID NO: 271). Inparticularly preferred embodiments, the polymeric carrier compoundconsists of a (R₁₂C)—(R₁₂C) dimer, a (WR₁₂C)—(WR₁₂C) dimer, or a(CR₁₂)—(CR₁₂C)—(CR₁₂) trimer, wherein the individual peptide elements inthe dimer (e.g. (WR12C)), or the trimer (e.g. (CR12)), are connected via—SH groups.

In a preferred embodiment of the second aspect, at least one nucleicacid (e.g. DNA or RNA), preferably the at least one RNA is complexed orassociated with a polyethylene glycol/peptide polymer comprisingHO-PEG5000-S—(S—CHHHHHHRRRRHHHHHHC—S-)7-S-PEG5000-OH (SEQ ID NO: 272 aspeptide monomer), HO-PEG5000-S—(S—CHHHHHHRRRRHHHHHHC—S-)4-S-PEG5000-OH(SEQ ID NO: 272 as peptide monomer),HO-PEG5000-S—(S—CGHHHHHRRRRHHHHHGC—S-)7-S-PEG5000-OH (SEQ ID NO: 273 aspeptide monomer) and/or a polyethylene glycol/peptide polymer comprisingHO-PEG5000-S—(S—CGHHHHHRRRRHHHHHGC—S-)4-S-PEG5000-OH (SEQ ID NO: 273 ofthe peptide monomer).

In other embodiments, the composition comprises at least one nucleicacid (e.g. DNA or RNA), wherein the at least one nucleic acid,preferably the at least one RNA is complexed or associated withpolymeric carriers and, optionally, with at least one lipid component asdescribed in WO2017/212008A1, WO2017/212006A1, WO2017/212007A1, andWO2017/212009A1. In this context, the disclosures of WO2017/212008A1,WO2017/212006A1, WO2017/212007A1, and WO2017/212009A1 are herewithincorporated by reference.

In a particularly preferred embodiment, the polymeric carrier (of thefirst and/or second component) is a peptide polymer, preferably apolyethylene glycol/peptide polymer as defined above, and a lipidcomponent, preferably a lipidoid component.

A lipidoid (or lipidoit) is a lipid-like compound, i.e. an amphiphiliccompound with lipid-like physical properties. The lipidoid is preferablya compound, which comprises two or more cationic nitrogen atoms and atleast two lipophilic tails. In contrast to many conventional cationiclipids, the lipidoid may be free of a hydrolysable linking group, inparticular linking groups comprising hydrolysable ester, amide orcarbamate groups. The cationic nitrogen atoms of the lipidoid may becationisable or permanently cationic, or both types of cationicnitrogens may be present in the compound. In the context of the presentinvention, the term lipid is considered to also encompass lipidoids.

In some embodiments of the inventions, the lipidoid may comprise a PEGmoiety.

In preferred embodiments, the at least one nucleic acid (e.g. DNA orRNA), preferably the at least one RNA is complexed or associated with apolymeric carrier, preferably with a polyethylene glycol/peptide polymeras defined above, and a lipidoid component.

Suitably, the lipidoid is cationic, which means that it is cationisableor permanently cationic. In one embodiment, the lipidoid iscationisable, i.e. it comprises one or more cationisable nitrogen atoms,but no permanently cationic nitrogen atoms. In another embodiment, atleast one of the cationic nitrogen atoms of the lipidoid is permanentlycationic. Optionally, the lipidoid comprises two permanently cationicnitrogen atoms, three permanently cationic nitrogen atoms, or even fouror more permanently cationic nitrogen atoms.

In a preferred embodiment, the lipidoid component may be any oneselected from the lipidoids of the lipidoids provided in the table ofpage 50-54 of published PCT patent application WO2017/212009A1, thespecific lipidoids provided in said table, and the specific disclosurerelating thereto herewith incorporated by reference.

In preferred embodiments, the lipidoid component may be any one selectedfrom 3-C12-OH, 3-C12-OH-cat, 3-C12-amide, 3-C12-amide monomethyl,3-C12-amide dimethyl, RevPEG(10)-3-C12-OH, RevPEG(10)-DLin-pAbenzoic,3C12amide-TMA cat., 3C12amide-DMA, 3C12amide-NH2, 3C12amide-OH,3C12Ester-OH, 3C12 Ester-amin, 3C12Ester-DMA, 2C12Amid-DMA,3C12-lin-amid-DMA, 2C12-sperm-amid-DMA, or 3C12-sperm-amid-DMA (seetable of published PCT patent application WO2017/212009A1 (pages50-54)). Particularly preferred are 3-C12-OH or 3-C12-OH-cat.

In preferred embodiments, the polyethylene glycol/peptide polymercomprising a lipidoid as specified above (e.g. 3-C12-OH or3-C12-OH-cat), is used to complex the at least one nucleic acid to formcomplexes having an N/P ratio from about 0.1 to about 20, or from about0.2 to about 15, or from about 2 to about 15, or from about 2 to about12, wherein the N/P ratio is defined as the mole ratio of the nitrogenatoms of the basic groups of the cationic peptide or polymer to thephosphate groups of the nucleic acid. In that context, the disclosure ofpublished PCT patent application WO2017/212009A1, in particular claims 1to 10 of WO2017/212009A1, and the specific disclosure relating theretois herewith incorporated by reference.

Further suitable lipidoids may be derived from published PCT patentapplication WO2010/053572. In particular, lipidoids derivable fromclaims 1 to 297 of published PCT patent application WO2010/053572 may beused in the context of the invention, e.g. incorporated into the peptidepolymer as described herein, or e.g. incorporated into the lipidnanoparticle (as described below). Accordingly, claims 1 to 297 ofpublished PCT patent application WO2010/053572, and the specificdisclosure relating thereto, is herewith incorporated by reference.

Encapsulation/Complexation in LNPs:

In preferred embodiments of the second aspect, the at least one nucleicacid (e.g. DNA or RNA), preferably the at least one RNA, and optionallythe at least one further nucleic acid, is complexed, encapsulated,partially encapsulated, or associated with one or more lipids (e.g.cationic lipids and/or neutral lipids), thereby forming lipid-basedcarriers such as liposomes, lipid nanoparticles (LNPs), lipoplexes,and/or nanoliposomes.

The liposomes, lipid nanoparticles (LNPs), lipoplexes, and/ornanoliposomes—incorporated nucleic acid (e.g. DNA or RNA) may becompletely or partially located in the interior space of the liposomes,lipid nanoparticles (LNPs), lipoplexes, and/or nanoliposomes, within thelipid layer/membrane, or associated with the exterior surface of thelipid layer/membrane. The incorporation of a nucleic acid intoliposomes/LNPs is also referred to herein as “encapsulation” wherein thenucleic acid, e.g. the RNA is entirely contained within the interiorspace of the liposomes, lipid nanoparticles (LNPs), lipoplexes, and/ornanoliposomes. The purpose of incorporating nucleic acid into liposomes,lipid nanoparticles (LNPs), lipoplexes, and/or nanoliposomes is toprotect the nucleic acid, preferably RNA from an environment which maycontain enzymes or chemicals or conditions that degrade nucleic acidand/or systems or receptors that cause the rapid excretion of thenucleic acid. Moreover, incorporating nucleic acid, preferably RNA intoliposomes, lipid nanoparticles (LNPs), lipoplexes, and/or nanoliposomesmay promote the uptake of the nucleic acid, and hence, may enhance thetherapeutic effect of the nucleic acid, e.g. the RNA encoding antigenicSARS-CoV-2 (nCoV-2019) proteins. Accordingly, incorporating a nucleicacid, e.g. RNA or DNA, into liposomes, lipid nanoparticles (LNPs),lipoplexes, and/or nanoliposomes may be particularly suitable for acoronavirus vaccine (e.g. a SARS-CoV-2 vaccine), e.g. for intramuscularand/or intradermal administration.

In this context, the terms “complexed” or “associated” refer to theessentially stable combination of nucleic acid with one or more lipidsinto larger complexes or assemblies without covalent binding.

The term “lipid nanoparticle”, also referred to as “LNP”, is notrestricted to any particular morphology, and include any morphologygenerated when a cationic lipid and optionally one or more furtherlipids are combined, e.g. in an aqueous environment and/or in thepresence of a nucleic acid, e.g. an RNA. For example, a liposome, alipid complex, a lipoplex and the like are within the scope of a lipidnanoparticle (LNP).

Liposomes, lipid nanoparticles (LNPs), lipoplexes, and/or nanoliposomescan be of different sizes such as, but not limited to, a multilamellarvesicle (MLV) which may be hundreds of nanometers in diameter and maycontain a series of concentric bilayers separated by narrow aqueouscompartments, a small unicellular vesicle (SUV) which may be smallerthan 50 nm in diameter, and a large unilamellar vesicle (LUV) which maybe between 50 nm and 500 nm in diameter.

LNPs of the invention are suitably characterized as microscopic vesicleshaving an interior aqua space sequestered from an outer medium by amembrane of one or more bilayers. Bilayer membranes of LNPs aretypically formed by amphiphilic molecules, such as lipids of syntheticor natural origin that comprise spatially separated hydrophilic andhydrophobic domains. Bilayer membranes of the liposomes can also beformed by amphiphilic polymers and surfactants (e.g., polymerosomes,niosomes, etc.). In the context of the present invention, an LNPtypically serves to transport the at least one nucleic acid, preferablythe at least one RNA to a target tissue.

Accordingly, in preferred embodiments of the second aspect, the at leastone nucleic acid, preferably the at least one RNA is complexed with oneor more lipids thereby forming lipid nanoparticles (LNP). Preferably,said LNP is particularly suitable for intramuscular and/or intradermaladministration. LNPs typically comprise a cationic lipid and one or moreexcipients selected from neutral lipids, charged lipids, steroids andpolymer conjugated lipids (e.g. PEGylated lipid). The nucleic acid (e.g.RNA, DNA) may be encapsulated in the lipid portion of the LNP or anaqueous space enveloped by some or the entire lipid portion of the LNP.The nucleic acid (e.g. RNA, DNA) or a portion thereof may also beassociated and complexed with the LNP. An LNP may comprise any lipidcapable of forming a particle to which the nucleic acids are attached,or in which the one or more nucleic acids are encapsulated. Preferably,the LNP comprising nucleic acids comprises one or more cationic lipids,and one or more stabilizing lipids. Stabilizing lipids include neutrallipids and PEGylated lipids.

Preferably, the LNP comprises

-   -   (i) at least one cationic lipid;    -   (ii) at least one neutral lipid;    -   (iii) at least one steroid or steroid analogue, preferably        cholesterol; and    -   (iv) at least one polymer conjugated lipid, preferably a        PEG-lipid;    -   wherein (i) to (iv) are in a molar ratio of about 20-60%        cationic lipid, 5-25% neutral lipid, 25-55% sterol, and 0.5-15%        polymer conjugated lipid.

The cationic lipid of an LNP may be cationisable, i.e. it becomesprotonated as the pH is lowered below the pK of the ionizable group ofthe lipid, but is progressively more neutral at higher pH values. At pHvalues below the pK, the lipid is then able to associate with negativelycharged nucleic acids. In certain embodiments, the cationic lipidcomprises a zwitterionic lipid that assumes a positive charge on pHdecrease.

Such lipids include, but are not limited to, DSDMA,N,N-dioleyl-N,N-dimethylammonium chloride (DODAC),N,N-distearyl-N,N-dimethylammonium bromide (DDAB), 1,2-dioleoyltrimethylammonium propane chloride (DOTAP) (also known asN-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride and1,2-Dioleyloxy-3-trimethylaminopropane chloride salt),N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA),N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), ckk-E12, ckk,1,2-DiLinoleyloxy-N,N-dimethylaminopropane (DLinDMA),1,2-Dilinolenyloxy-N, N-dimethylaminopropane (DLenDMA),1,2-di-y-linolenyloxy-N,N-dimethylaminopropane (γ-DLenDMA), 98N12-5,1,2-Dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP),1,2-Dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC),1,2-Dilinoleyoxy-3-morpholinopropane (DLin-MA),1,2-Dilinoleoyl-3-dimethylaminopropane (DLinDAP),1,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA),1-Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP),1,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.CI),ICE (Imidazol-based), HGT5000, HGT5001, DMDMA, CLinDMA, CpLinDMA, DMOBA,DOcarbDAP, DLincarbDAP, DLinCDAP, KLin-K-DMA, DLin-K-XTC2-DMA, XTC(2,2-Dilinoleyl-4-dimethylaminoethyl[1,3]-dioxolane) HGT4003,1,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.CI),1,2-Dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), or3-(N,N-Dilinoleylamino)-1,2-propanediol (DLinAP),3-(N,N-Dioleylamino)-1,2-propanedio (DOAP),1,2-Dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DM A),2,2-Dilinoleyl-4-dimethylaminomethyl[1,3]-dioxolane (DLin-K-DMA) oranalogs thereof,(3aR,5s,6aS)—N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine,(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate(MC3), ALNY-100((3aR,5s,6aS)—N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine)),1,1′-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazanediyl)didodecan-2-ol(C12-200), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)[1,3]-dioxolane(DLin-K-C2-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl[1,3]-dioxolane(DLin-K-DMA), NC98-5(4,7,13-tris(3-oxo-3-(undecylamino)propyl)-N1,N16-diundecyl-4,7,10,13-tetraazahexadecane-1,16-diamide),(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl4-(dimethylamino)butanoate (DLin-M-C3-DMA),3-((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yloxy)-N,N-dimethylpropan-1-amine(MC3 Ether),4-((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yloxy)-N,N-dimethylbutan-1-amine(MC4 Ether), LIPOFECTIN® (commercially available cationic liposomescomprising DOTMA and 1,2-dioleoyl-sn-3phosphoethanolamine (DOPE), fromGIBCO/BRL, Grand Island, N.Y.); LIPOFECTAMINE® (commercially availablecationic liposomes comprisingN-(1-(2,3dioleyloxy)propyl)-N-(2-(sperminecarboxamido)ethyl)-N,N-dimethylammoniumtrifluoroacetate (DOSPA) and (DOPE), from GIBCO/BRL); and TRANSFECTAM®(commercially available cationic lipids comprisingdioctadecylamidoglycyl carboxyspermine (DOGS) in ethanol from PromegaCorp., Madison, Wis.) or any combination of any of the foregoing.Further suitable cationic lipids for use in the compositions and methodsof the invention include those described in international patentpublications WO2010/053572 (and particularly, CI 2-200 described atparagraph [00225]) and WO2012/170930, both of which are incorporatedherein by reference, HGT4003, HGT5000, HGTS001, HGT5001, HGT5002 (seeUS20150140070A1).

In embodiments, the cationic lipid may be an amino lipid.

Representative amino lipids include, but are not limited to,1,2-dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC),1,2-dilinoleyoxy-3morpholinopropane (DLin-MA),1,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP),1,2-dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA),1-linoleoyl-2-linoleyloxy-3dimethylaminopropane (DLin-2-DMAP),1,2-dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.CI),1,2-dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.CI),1,2-dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ),3-(N,Ndilinoleylamino)-1,2-propanediol (DLinAP),3-(N,N-dioleylamino)-1,2-propanediol (DOAP),1,2-dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA),and 2,2-dilinoleyl-4-dimethylaminomethyl[1,3]-dioxolane (DLin-K-DMA),2,2-dilinoleyl-4-(2-dimethylaminoethyl)[1,3]-dioxolane (DLin-KC2-DMA);dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA); MC3(US20100324120).

In embodiments, the cationic lipid may an aminoalcohol lipidoid.

Aminoalcohol lipidoids which may be used in the present invention may beprepared by the methods described in U.S. Pat. No. 8,450,298, hereinincorporated by reference in its entirety. Suitable (ionizable) lipidscan also be the compounds as disclosed in Tables 1, 2 and 3 and asdefined in claims 1-24 of WO2017/075531A1, hereby incorporated byreference.

In another embodiment, suitable lipids can also be the compounds asdisclosed in WO2015/074085A1 (i.e. ATX-001 to ATX-032 or the compoundsas specified in claims 1-26), U.S. Appl. Nos. 61/905,724 and Ser. No.15/614,499 or U.S. Pat. Nos. 9,593,077 and 9,567,296 hereby incorporatedby reference in their entirety.

In other embodiments, suitable cationic lipids can also be the compoundsas disclosed in WO2017/117530A1 (i.e. lipids 13, 14, 15, 16, 17, 18, 19,20, or the compounds as specified in the claims), hereby incorporated byreference in its entirety.

In preferred embodiments, ionizable or cationic lipids may also beselected from the lipids disclosed in WO2018/078053A1 (i.e. lipidsderived from formula I, II, and III of WO2018/078053A1, or lipids asspecified in claims 1 to 12 of WO2018/078053A1), the disclosure ofWO2018/078053A1 hereby incorporated by reference in its entirety. Inthat context, lipids disclosed in Table 7 of WO2018/078053A1 (e.g.lipids derived from formula I-1 to 1-41) and lipids disclosed in Table 8of WO2018/078053A1 (e.g. lipids derived from formula II-1 to 11-36) maybe suitably used in the context of the invention. Accordingly, formulaI-1 to formula I-41 and formula II-1 to formula II-36 ofWO2018/078053A1, and the specific disclosure relating thereto, areherewith incorporated by reference.

In preferred embodiments, cationic lipids may be derived from formulaIII of published PCT patent application WO2018/078053A1. Accordingly,formula III of WO2018/078053A1, and the specific disclosure relatingthereto, are herewith incorporated by reference.

In particularly preferred embodiments, the at least one nucleic acid(e.g. DNA or RNA), preferably the at least one RNA of the composition iscomplexed with one or more lipids thereby forming LNPs, wherein thecationic lipid of the LNP is selected from structures III-1 to III-36 ofTable 9 of published PCT patent application WO2018/078053A1.Accordingly, formula III-1 to III-36 of WO2018/078053A1, and thespecific disclosure relating thereto, are herewith incorporated byreference.

In particularly preferred embodiment of the second aspect, the at leastone nucleic acid (e.g. DNA or RNA), preferably the at least one RNA iscomplexed with one or more lipids thereby forming LNPs, wherein the LNPscomprises a cationic lipid according to formula III-3:

The lipid of formula III-3 as suitably used herein has the chemical term((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate),also referred to as ALC-0315.

In certain embodiments, the cationic lipid as defined herein, morepreferably cationic lipid compound 111-3, is present in the LNP in anamount from about 30 to about 95 mole percent, relative to the totallipid content of the LNP. If more than one cationic lipid isincorporated within the LNP, such percentages apply to the combinedcationic lipids.

In embodiments, the cationic lipid is present in the LNP in an amountfrom about 30 to about 70 mole percent. In one embodiment, the cationiclipid is present in the LNP in an amount from about 40 to about 60 molepercent, such as about 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,52, 53, 54, 55, 56, 57, 58, 59 or 60 mole percent, respectively. Inembodiments, the cationic lipid is present in the LNP in an amount fromabout 47 to about 48 mole percent, such as about 47.0, 47.1, 47.2, 47.3,47.4, 47.5, 47.6, 47.7, 47.8, 47.9, 50.0 mole percent, respectively,wherein 47.7 mole percent are particularly preferred.

In some embodiments, the cationic lipid is present in a ratio of fromabout 20 mol % to about 70 or 75 mol % or from about 45 to about 65 mol% or about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or about 70 mol % ofthe total lipid present in the LNP. In further embodiments, the LNPscomprise from about 25% to about 75% on a molar basis of cationic lipid,e.g., from about 20 to about 70%, from about 35 to about 65%, from about45 to about 65%, about 60%, about 57.5%, about 57.1%, about 50% or about40% on a molar basis (based upon 100% total moles of lipid in the lipidnanoparticle). In some embodiments, the ratio of cationic lipid tonucleic acid (e.g. coding RNA or DNA) is from about 3 to about 15, suchas from about 5 to about 13 or from about 7 to about 11.

Other suitable (cationic or ionizable) lipids are disclosed inWO2009/086558, WO2009/127060, WO2010/048536, WO2010/054406,WO2010/088537, WO2010/129709, WO2011/153493, WO 2013/063468,US2011/0256175, US2012/0128760, US2012/0027803, U.S. Pat. No. 8,158,601,WO2016/118724, WO2016/118725, WO2017/070613, WO2017/070620,WO2017/099823, WO2012/040184, WO2011/153120, WO2011/149733,WO2011/090965, WO2011/043913, WO2011/022460, WO2012/061259,WO2012/054365, WO2012/044638, WO2010/080724, WO2010/21865,WO2008/103276, WO2013/086373, WO2013/086354, U.S. Pat. Nos. 7,893,302,7,404,969, 8,283,333, 8,466,122 and 8,569,256 and US Patent PublicationNo. US2010/0036115, US2012/0202871, US2013/0064894, US2013/0129785,US2013/0150625, US2013/0178541, US2013/0225836, US2014/0039032 andWO2017/112865. In that context, the disclosures of WO2009/086558,WO2009/127060, WO2010/048536, WO2010/054406, WO2010/088537,WO2010/129709, WO2011/153493, WO 2013/063468, U52011/0256175,US2012/0128760, US2012/0027803, U.S. Pat. No. 8,158,601, WO2016/118724,WO2016/118725, WO2017/070613, WO2017/070620, WO2017/099823,WO2012/040184, WO2011/153120, WO2011/149733, WO2011/090965,WO2011/043913, WO2011/022460, WO2012/061259, WO2012/054365,WO2012/044638, WO2010/080724, WO2010/21865, WO2008/103276,WO2013/086373, WO2013/086354, U.S. Pat. Nos. 7,893,302, 7,404,969,8,283,333, 8,466,122 and 8,569,256 and US Patent Publication No.US2010/0036115, US2012/0202871, US2013/0064894, US2013/0129785,US2013/0150625, US2013/0178541, US2013/0225836 and US2014/0039032 andWO2017/112865 specifically relating to (cationic) lipids suitable forLNPs are incorporated herewith by reference.

In embodiments, amino or cationic lipids as defined herein have at leastone protonatable or deprotonatable group, such that the lipid ispositively charged at a pH at or below physiological pH (e.g. pH 7.4),and neutral at a second pH, preferably at or above physiological pH. Itwill, of course, be understood that the addition or removal of protonsas a function of pH is an equilibrium process, and that the reference toa charged or a neutral lipid refers to the nature of the predominantspecies and does not require that all of lipids have to be present inthe charged or neutral form. Lipids having more than one protonatable ordeprotonatable group, or which are zwitterionic, are not excluded andmay likewise suitable in the context of the present invention. In someembodiments, the protonatable lipids have a pKa of the protonatablegroup in the range of about 4 to about 11, e.g., a pKa of about 5 toabout 7.

LNPs can comprise two or more (different) cationic lipids as definedherein. Cationic lipids may be selected to contribute to differentadvantageous properties. For example, cationic lipids that differ inproperties such as amine pKa, chemical stability, half-life incirculation, half-life in tissue, net accumulation in tissue, ortoxicity can be used in the LNP. In particular, the cationic lipids canbe chosen so that the properties of the mixed-LNP are more desirablethan the properties of a single-LNP of individual lipids.

The amount of the permanently cationic lipid or lipidoid may be selectedtaking the amount of the nucleic acid cargo into account. In oneembodiment, these amounts are selected such as to result in an N/P ratioof the nanoparticle(s) or of the composition in the range from about 0.1to about 20. In this context, the N/P ratio is defined as the mole ratioof the nitrogen atoms (“N”) of the basic nitrogen-containing groups ofthe lipid or lipidoid to the phosphate groups (“P”) of the nucleic acidwhich is used as cargo. The N/P ratio may be calculated on the basisthat, for example, 1 ug RNA typically contains about 3 nmol phosphateresidues, provided that the RNA exhibits a statistical distribution ofbases. The “N”-value of the lipid or lipidoid may be calculated on thebasis of its molecular weight and the relative content of permanentlycationic and—if present—cationisable groups.

In vivo characteristics and behavior of LNPs can be modified by additionof a hydrophilic polymer coating, e.g. polyethylene glycol (PEG), to theLNP surface to confer steric stabilization. Furthermore, LNPs can beused for specific targeting by attaching ligands (e.g. antibodies,peptides, and carbohydrates) to its surface or to the terminal end ofthe attached PEG chains (e.g. via PEGylated lipids or PEGylatedcholesterol).

In some embodiments, the LNPs comprise a polymer conjugated lipid. Theterm “polymer conjugated lipid” refers to a molecule comprising both alipid portion and a polymer portion. An example of a polymer conjugatedlipid is a PEGylated lipid. The term “PEGylated lipid” refers to amolecule comprising both a lipid portion and a polyethylene glycolportion. PEGylated lipids are known in the art and include1-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-s-DMG)and the like.

A polymer conjugated lipid as defined herein, e.g. a PEG-lipid, mayserve as an aggregation reducing lipid.

In certain embodiments, the LNP comprises a stabilizing-lipid which is apolyethylene glycol-lipid (PEGylated lipid). Suitable polyethyleneglycol-lipids include PEG-modified phosphatidylethanolamine,PEG-modified phosphatidic acid, PEG-modified ceramides (e.g. PEG-CerC14or PEG-CerC20), PEG-modified dialkylamines, PEG-modifieddiacylglycerols, PEG-modified dialkylglycerols. Representativepolyethylene glycol-lipids include PEG-c-DOMG, PEG-c-DMA, and PEG-s-DMG.In one embodiment, the polyethylene glycol-lipid is N-[(methoxypoly(ethylene glycol)2000)carbamyI]-1,2-dimyristyloxlpropyl-3-amine(PEG-c-DMA). In a preferred embodiment, the polyethylene glycol-lipid isPEG-2000-DMG. In one embodiment, the polyethylene glycol-lipid isPEG-c-DOMG). In other embodiments, the LNPs comprise a PEGylateddiacylglycerol (PEG-DAG) such as1-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-DMG), aPEGylated phosphatidylethanoloamine (PEG-PE), a PEG succinatediacylglycerol (PEG-S-DAG) such as4-O-(2′,3′-di(tetradecanoyloxy)propyl-1-O-(ω-methoxy(polyethoxy)ethyl)butanedioate(PEG-S-DMG), a PEGylated ceramide (PEG-cer), or a PEGdialkoxypropylcarbamate such asω-methoxy(polyethoxy)ethyl-N-(2,3di(tetradecanoxy)propyl)carbamate or2,3-di(tetradecanoxy)propyl-N-(ω-methoxy(polyethoxy)ethyl)carbamate.

In preferred embodiments, the PEGylated lipid is preferably derived fromformula (IV) of published PCT patent application WO2018/078053A1.Accordingly, PEGylated lipids derived from formula (IV) of published PCTpatent application WO2018/078053A1, and the respective disclosurerelating thereto, are herewith incorporated by reference.

In a particularly preferred embodiments, the at least one nucleic acid(e.g. RNA or DNA) of the composition is complexed with one or morelipids thereby forming LNPs, wherein the LNP comprises a PEGylatedlipid, wherein the PEG lipid is preferably derived from formula (IVa) ofpublished PCT patent application WO2018/078053A1.

Accordingly, PEGylated lipid derived from formula (IVa) of published PCTpatent application WO2018/078053A1, and the respective disclosurerelating thereto, is herewith incorporated by reference.

In a particularly preferred embodiment, the at least one nucleic acid,preferably the at least one RNA is complexed with one or more lipidsthereby forming lipid nanoparticles (LNP), wherein the LNP comprises aPEGylated lipid/PEG lipid. Preferably, said PEG lipid is of formula(IVa):

wherein n has a mean value ranging from 30 to 60, such as about 30±2,32±2, 34±2, 36±2, 38±2, 40±2, 42±2, 44±2, 46±2, 48±2, 50±2, 52±2, 54±2,56±2, 58±2, or 60±2. In a most preferred embodiment n is about 49. Infurther preferred aspects said PEG lipid is of formula (IVa) wherein nis an integer selected such that the average molecular weight of the PEGlipid is about 2000 g/mol to about 3000 g/mol or about 2300 g/mol toabout 2700 g/mol, even more preferably about 2500 g/mol.

The lipid of formula IVa as suitably used herein has the chemical term2[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide, also referredto as ALC-0159.

Further examples of PEG-lipids suitable in that context are provided inUS2015/0376115A1 and WO2015/199952, each of which is incorporated byreference in its entirety.

In some embodiments, LNPs include less than about 3, 2, or 1 molepercent of PEG or PEG-modified lipid, based on the total moles of lipidin the LNP. In further embodiments, LNPs comprise from about 0.1% toabout 20% of the PEG-modified lipid on a molar basis, e.g., about 0.5 toabout 10%, about 0.5 to about 5%, about 10%, about 5%, about 3.5%, about3%, about 2,5%, about 2%, about 1.5%, about 1%, about 0.5%, or about0.3% on a molar basis (based on 100% total moles of lipids in the LNP).In preferred embodiments, LNPs comprise from about 1.0% to about 2.0% ofthe PEG-modified lipid on a molar basis, e.g., about 1.2 to about 1.9%,about 1.2 to about 1.8%, about 1.3 to about 1.8%, about 1.4 to about1.8%, about 1.5 to about 1.8%, about 1.6 to about 1.8%, in particularabout 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%,most preferably 1.7% (based on 100% total moles of lipids in the LNP).In various embodiments, the molar ratio of the cationic lipid to thePEGylated lipid ranges from about 100:1 to about 25:1.

In preferred embodiments, the LNP comprises one or more additionallipids, which stabilize the formation of particles during theirformation or during the manufacturing process (e.g. neutral lipid and/orone or more steroid or steroid analogue).

In preferred embodiments of the second aspect, the at least one nucleicacid, preferably the at least one RNA is complexed with one or morelipids thereby forming lipid nanoparticles (LNP), wherein the LNPcomprises one or more neutral lipid and/or one or more steroid orsteroid analogue.

Suitable stabilizing lipids include neutral lipids and anionic lipids.The term “neutral lipid” refers to any one of a number of lipid speciesthat exist in either an uncharged or neutral zwitterionic form atphysiological pH. Representative neutral lipids includediacylphosphatidylcholines, diacylphosphatidylethanolamines, ceramides,sphingomyelins, dihydro sphingomyelins, cephalins, and cerebrosides.

In embodiments of the second aspect, the LNP comprises one or moreneutral lipids, wherein the neutral lipid is selected from the groupcomprising distearoylphosphatidylcholine (DSPC),dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine(DPPC), dioleoylphosphatidylglycerol (DOPG),dipalmitoylphosphatidylglycerol (DPPG),dioleoyl-phosphatidylethanolamine (DOPE),palmitoyloleoylphosphatidylcholine (POPC),palmitoyloleoyl-phosphatidylethanolamine (POPE) anddioleoyl-phosphatidylethanolamine4-(N-maleimidomethyl)-cyclohexane-1carboxylate (DOPE-mal), dipalmitoylphosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE),distearoyl-phosphatidylethanolamine (DSPE), 16-O-monomethyl PE,16-O-dimethyl PE, 18-1-trans PE, 1-stearioyl-2-oleoylphosphatidyethanolamine (SOPE), and 1,2-dielaidoyl-sn-glycero-3-phophoethanolamine(transDOPE), or mixtures thereof.

In some embodiments, the LNPs comprise a neutral lipid selected fromDSPC, DPPC, DMPC, DOPC, POPC, DOPE and SM. In various embodiments, themolar ratio of the cationic lipid to the neutral lipid ranges from about2:1 to about 8:1.

In preferred embodiments, the neutral lipid is1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). The molar ratio ofthe cationic lipid to DSPC may be in the range from about 2:1 to about8:1.

In preferred embodiments, the steroid is cholesterol. The molar ratio ofthe cationic lipid to cholesterol may be in the range from about 2:1 toabout 1:1. In some embodiments, the cholesterol may be PEGylated.

The sterol can be about 10 mol % to about 60 mol % or about 25 mol % toabout 40 mol % of the lipid particle. In one embodiment, the sterol isabout 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or about 60 mol % of thetotal lipid present in the lipid particle. In another embodiment, theLNPs include from about 5% to about 50% on a molar basis of the sterol,e.g., about 15% to about 45%, about 20% to about 40%, about 48%, about40%, about 38.5%, about 35%, about 34.4%, about 31.5% or about 31% on amolar basis (based upon 100% total moles of lipid in the lipidnanoparticle).

Preferably, lipid nanoparticles (LNPs) comprise: (a) the at least onenucleic acid, preferably the at least one RNA of the first aspect, (b) acationic lipid, (c) an aggregation reducing agent (such as polyethyleneglycol (PEG) lipid or PEG-modified lipid), (d) optionally a non-cationiclipid (such as a neutral lipid), and (e) optionally, a sterol.

In some embodiments, the cationic lipids (as defined above),non-cationic lipids (as defined above), cholesterol (as defined above),and/or PEG-modified lipids (as defined above) may be combined at variousrelative molar ratios. For example, the ratio of cationic lipid tonon-cationic lipid to cholesterol-based lipid to PEGylated lipid may bebetween about 30-60:20-35:20-30:1-15, or at a ratio of about 40:30:25:5,50:25:20:5, 50:27:20:3, 40:30:20:10, 40:32:20:8, 40:32:25:3 or40:33:25:2, or at a ratio of about 50:25:20:5, 50:20:25:5, 50:27:20:3,40:30:20:10, 40:30:25:5 or 40:32:20:8, 40:32:25:3 or 40:33:25:2,respectively.

In some embodiments, the LNPs comprise a lipid of formula (III), the atleast one nucleic acid, preferably the at least one RNA as definedherein, a neutral lipid, a steroid and a PEGylated lipid. In preferredembodiments, the lipid of formula (III) is lipid compound III-3(ALC-0315), the neutral lipid is DSPC, the steroid is cholesterol, andthe PEGylated lipid is the compound of formula (IVa) (ALC-0159).

In a preferred embodiment of the second aspect, the LNP consistsessentially of (i) at least one cationic lipid; (ii) a neutral lipid;(iii) a sterol, e.g., cholesterol; and (iv) a PEG-lipid, e.g. PEG-DMG orPEG-cDMA, in a molar ratio of about 20-60% cationic lipid:5-25% neutrallipid:25-55% sterol; 0.5-15% PEG-lipid.

In particularly preferred embodiments, the at least one nucleic acid,preferably the at least one RNA is complexed with one or more lipidsthereby forming lipid nanoparticles (LNP), wherein the LNP comprises

-   -   (i) at least one cationic lipid as defined herein, preferably a        lipid of formula (III), more preferably lipid III-3 (ALC-0315);    -   (ii) at least one neutral lipid as defined herein, preferably        1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC);    -   (iii) at least one steroid or steroid analogue as defined        herein, preferably cholesterol; and    -   (iv) at least one PEG-lipid as defined herein, e.g. PEG-DMG or        PEG-cDMA, preferably a PEGylated lipid that is or is derived        from formula (IVa) (ALC-0159).

In particularly preferred embodiments, the at least one nucleic acid,preferably the at least one RNA is complexed with one or more lipidsthereby forming lipid nanoparticles (LNP), wherein the LNP comprises (i)to (iv) in a molar ratio of about 20-60% cationic lipid: 5-25% neutrallipid: 25-55% sterol; 0.5-15% PEG-lipid.

In one preferred embodiment, the lipid nanoparticle comprises: acationic lipid with formula (III) and/or PEG lipid with formula (IV),optionally a neutral lipid, preferably1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and optionally asteroid, preferably cholesterol, wherein the molar ratio of the cationiclipid to DSPC is optionally in the range from about 2:1 to 8:1, whereinthe molar ratio of the cationic lipid to cholesterol is optionally inthe range from about 2:1 to 1:1.

In a particular preferred embodiment, the composition of the secondaspect comprising the at least one nucleic acid, preferably the at leastone RNA, comprises lipid nanoparticles (LNPs), which have a molar ratioof approximately 50:10:38.5:1.5, preferably 47.5:10:40.8:1.7 or morepreferably 47.4:10:40.9:1.7 (i.e. proportion (mol %) of cationic lipid(preferably lipid III-3 (ALC-0315)), DSPC, cholesterol and PEG-lipid(preferably PEG-lipid of formula (IVa) with n=49, even more preferablyPEG-lipid of formula (IVa) with n=45 (ALC-0159)); solubilized inethanol).

Most preferably, the composition of the second aspect comprises at leastone nucleic acid, preferably RNA, which is identical or at least 70%,80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identical to a nucleic acid sequence of SEQ ID NO: 163formulated in lipid nanoparticles (LNPs), which have a molar ratio ofapproximately 50:10:38.5:1.5, preferably 47.5:10:40.8:1.7 or morepreferably 47.4:10:40.9:1.7 proportion (mol %) of cationic lipid III-3(ALC-0315), DSPC, cholesterol and PEG-lipid of formula (IVa) (with n=49or with n=45 (ALC-0159)). In this preferred embodiment the nucleic acid,preferably mRNA is not chemically modified.

In another most preferred embodiment, the composition of the secondaspect comprises at least one nucleic acid, preferably RNA, which isidentical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acidsequence of SEQ ID NO: 149 formulated in lipid nanoparticles (LNPs),which have a molar ratio of approximately 50:10:38.5:1.5, preferably47.5:10:40.8:1.7 or more preferably 47.4:10:40.9:1.7 proportion (mol %)of cationic lipid III-3 (ALC-0315), DSPC, cholesterol and PEG-lipid offormula (IVa) (with n=49 or with n=45 (ALC-0159)). In this preferredembodiment the nucleic acid, preferably mRNA is not chemically modified.

In another most preferred embodiment, the composition of the secondaspect comprises at least one nucleic acid, preferably RNA, which isidentical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acidsequence of SEQ ID NO: 24837 formulated in lipid nanoparticles (LNPs),which have a molar ratio of approximately 50:10:38.5:1.5, preferably47.5:10:40.8:1.7 or more preferably 47.4:10:40.9:1.7 proportion (mol %)of cationic lipid III-3 (ALC-0315), DSPC, cholesterol and PEG-lipid offormula (IVa) (with n=49 or with n=45) (ALC-0159). In this preferredembodiment the nucleic acid, preferably mRNA is not chemically modified.

In a further preferred embodiment, the composition of the second aspectcomprises at least one nucleic acid, preferably RNA, which is identicalor at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence of SEQID NO: 23311, 23531, or 24851 formulated in lipid nanoparticles (LNPs),which have a molar ratio of approximately 50:10:38.5:1.5, preferably47.5:10:40.8:1.7 or more preferably 47.4:10:40.9:1.7 proportion (mol %)of cationic lipid III-3 (ALC-0315), DSPC, cholesterol and PEG-lipid offormula (IVa) (with n=49 or with n=45) (ALC-0159). In this preferredembodiment the nucleic acid, preferably mRNA is not chemically modified.

In a further preferred embodiment, the composition of the second aspectcomprises at least one nucleic acid, preferably RNA, which is identicalor at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence of SEQID NO: 23310, 23530, 23313, or 23533 formulated in lipid nanoparticles(LNPs), which have a molar ratio of approximately 50:10:38.5:1.5,preferably 47.5:10:40.8:1.7 or more preferably 47.4:10:40.9:1.7proportion (mol %) of cationic lipid III-3 (ALC-0315), DSPC, cholesteroland PEG-lipid of formula (IVa) (with n=49 or with n=45) (ALC-0159). Inthis preferred embodiment the nucleic acid, preferably mRNA is notchemically modified.

In embodiments where the composition is a multivalent composition asdefined above, the nucleic acid species (e.g. DNA or RNA), preferablyRNA species of the multivalent composition may be formulated separately,preferably formulated separately in liposomes or LNPs. Suitably, the RNAspecies of the multivalent composition are separately formulated in LNPswhich have a molar ratio of approximately 50:10:38.5:1.5, preferably47.5:10:40.8:1.7 or more preferably 47.4:10:40.9:1.7 proportion (mol %)of cationic lipid III-3 (ALC-0315), DSPC, cholesterol and PEG-lipid offormula (IVa) (with n=49 or with n=45). Nucleic acid species formultivalent compositions are preferably selected as defined above (seesection “Multivalent compositions of the invention”)

In embodiments where the composition is a multivalent composition asdefined above, the nucleic acid species (e.g. DNA or RNA), preferablyRNA species of the multivalent composition may be co-formulated,preferably co-formulated in liposomes or LNPs. Suitably, the RNA speciesof the multivalent composition are co-formulated in LNPs which have amolar ratio of approximately 50:10:38.5:1.5, preferably 47.5:10:40.8:1.7or more preferably 47.4:10:40.9:1.7 proportion (mol %) of cationic lipidIII-3 (ALC-0315), DSPC, cholesterol and PEG-lipid of formula (IVa) (withn=49 or with n=45).

Nucleic acid species for multivalent compositions are preferablyselected as defined above (see section “Multivalent compositions of theinvention”)

The total amount of nucleic acid in the lipid nanoparticles may vary andis defined depending on the e.g. nucleic acid to total lipid w/w ratio.In one embodiment of the invention the nucleic acid, in particular theRNA to total lipid ratio is less than 0.06 w/w, preferably between 0.03w/w and 0.04 w/w.

In some embodiments, the lipid nanoparticles (LNPs), which are composedof only three lipid components, namely imidazole cholesterol ester(ICE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and1,2-dimyristoyl-sn-glycerol, methoxypolyethylene glycol (DMG-PEG-2K).

In one embodiment, the lipid nanoparticle of the composition comprises acationic lipid, a steroid; a neutral lipid; and a polymer conjugatedlipid, preferably a pegylated lipid. Preferably, the polymer conjugatedlipid is a pegylated lipid or PEG-lipid. In a specific embodiment, lipidnanoparticles comprise a cationic lipid resembled by the cationic lipidCOATSOME® SS-EC (former name: SS-33/4PE-15; NOF Corporation, Tokyo,Japan), in accordance with the following formula

As described further below, those lipid nanoparticles are termed “GN01”.

Furthermore, in a specific embodiment, the GN01 lipid nanoparticlescomprise a neutral lipid being resembled by the structure1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPhyPE):

Furthermore, in a specific embodiment, the GN01 lipid nanoparticlescomprise a polymer conjugated lipid, preferably a pegylated lipid, being1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol 2000 (DMG-PEG2000) having the following structure:

As used in the art, “DMG-PEG 2000” is considered a mixture of 1,2-DMGPEG2000 and 1,3-DMG PEG2000 in ˜97:3 ratio.

Accordingly, GN01 lipid nanoparticles (GN01-LNPs) according to one ofthe preferred embodiments comprise a SS-EC cationic lipid, neutral lipidDPhyPE, cholesterol, and the polymer conjugated lipid (pegylated lipid)1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (PEG-DMG).

In a preferred embodiment, the GN01 LNPs comprise:

(a) cationic lipid SS-EC (former name: SS-33/4PE-15; NOF Corporation,Tokyo, Japan) at an amount of 45-65 mol %;

(b) cholesterol at an amount of 25-45 mol %;

(c) DPhyPE at an amount of 8-12 mol %; and

(d) PEG-DMG 2000 at an amount of 1-3 mol %;

each amount being relative to the total molar amount of all lipidicexcipients of the GN01 lipid nanoparticles.

In a further preferred embodiment, the GN01 lipid nanoparticles asdescribed herein comprises 59 mol % cationic lipid, 10 mol % neutrallipid, 29.3 mol % steroid and 1.7 mol % polymer conjugated lipid,preferably pegylated lipid. In a most preferred embodiment, the GN01lipid nanoparticles as described herein comprise 59 mol % cationic lipidSS-EC, 10 mol % DPhyPE, 29.3 mol % cholesterol and 1.7 mol % DMG-PEG2000.

The amount of the cationic lipid relative to that of the nucleic acid inthe GN01 lipid nanoparticle may also be expressed as a weight ratio(abbreviated f.e. “m/m”). For example, the GN01 lipid nanoparticlescomprise the at least one nucleic acid, preferably the at least one RNAat an amount such as to achieve a lipid to RNA weight ratio in the rangeof about 20 to about 60, or about 10 to about 50. In other embodiments,the ratio of cationic lipid to nucleic acid or RNA is from about 3 toabout 15, such as from about 5 to about 13, from about 4 to about 8 orfrom about 7 to about 11. In a very preferred embodiment of the presentinvention, the total lipid/RNA mass ratio is about 40 or 40, i.e. about40 or 40 times mass excess to ensure RNA encapsulation. Anotherpreferred RNA/lipid ratio is between about 1 and about 10, about 2 andabout 5, about 2 and about 4, or preferably about 3.

Further, the amount of the cationic lipid may be selected taking theamount of the nucleic acid cargo such as the RNA compound into account.In one embodiment, the N/P ratio can be in the range of about 1 to about50. In another embodiment, the range is about 1 to about 20, about 1 toabout 10, about 1 to about 5. In one preferred embodiment, these amountsare selected such as to result in an N/P ratio of the GN01 lipidnanoparticles or of the composition in the range from about 10 to about20. In a further very preferred embodiment, the N/P is 14 (i.e. 14 timesmol excess of positive charge to ensure nucleic acid encapsulation).

In a preferred embodiment, GN01 lipid nanoparticles comprise 59 mol %cationic lipid COATSOME® SS-EC (former name: SS-33/4PE-15 as apparentfrom the examples section; NOF Corporation, Tokyo, Japan), 29.3 mol %cholesterol as steroid, 10 mol % DPhyPE as neutral lipid/phospholipidand 1.7 mol % DMG-PEG 2000 as polymer conjugated lipid. A furtherinventive advantage connected with the use of DPhyPE is the highcapacity for fusogenicity due to its bulky tails, whereby it is able tofuse at a high level with endosomal lipids. For “GN01”, N/P (lipid tonucleic acid, e.g RNA mol ratio) preferably is 14 and total lipid/RNAmass ratio preferably is 40 (m/m).

In other embodiments, the at least one nucleic acid (e.g. DNA or RNA),preferably the at least one RNA is complexed with one or more lipidsthereby forming lipid nanoparticles (LNP), wherein the LNP comprises

-   -   I at least one cationic lipid;    -   Ii at least one neutral lipid;    -   Iii at least one steroid or steroid analogue; and    -   Iiii at least one PEG-lipid as defined herein,

wherein the cationic lipid is DLin-KC2-DMA (50 mol %) or DLin-MC3-DMA(50 mol %), the neutral lipid is DSPC (10 mol %), the PEG lipid isPEG-DOMG (1.5 mol %) and the structural lipid is cholesterol (38.5 mol%).

In other embodiments, the at least one nucleic acid (e.g. DNA or RNA),preferably the at least one RNA is complexed with one or more lipidsthereby forming lipid nanoparticles (LNP), wherein the LNP comprisesSS15/Chol/DOPE (or DOPC)/DSG-5000 at mol % 50/38.5/10/1.5.

In other embodiments, the nucleic acid of the invention may beformulated in liposomes, e.g. in liposomes as described inWO2019/222424, WO2019/226925, WO2019/232095, WO2019/232097, orWO2019/232208, the disclosure of WO2019/222424, WO2019/226925,WO2019/232095, WO2019/232097, or WO2019/232208 relating to liposomes orlipid-based carrier molecules herewith incorporated by reference.

In various embodiments, LNPs that suitably encapsulates the at least onenucleic acid of the invention have a mean diameter of from about 50 nmto about 200 nm, from about 60 nm to about 200 nm, from about 70 nm toabout 200 nm, from about 80 nm to about 200 nm, from about 90 nm toabout 200 nm, from about 90 nm to about 190 nm, from about 90 nm toabout 180 nm, from about 90 nm to about 170 nm, from about 90 nm toabout 160 nm, from about 90 nm to about 150 nm, from about 90 nm toabout 140 nm, from about 90 nm to about 130 nm, from about 90 nm toabout 120 nm, from about 90 nm to about 100 nm, from about 70 nm toabout 90 nm, from about 80 nm to about 90 nm, from about 70 nm to about80 nm, or about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm,70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, 150 nm, 160 nm, 170nm, 180 nm, 190 nm, or 200 nm and are substantially non-toxic. As usedherein, the mean diameter may be represented by the z-average asdetermined by dynamic light scattering as commonly known in the art.

The polydispersity index (PDI) of the nanoparticles is typically in therange of 0.1 to 0.5. In a particular embodiment, a PDI is below 0.2.Typically, the PDI is determined by dynamic light scattering.

In another preferred embodiment of the invention the lipid nanoparticleshave a hydrodynamic diameter in the range from about 50 nm to about 300nm, or from about 60 nm to about 250 nm, from about 60 nm to about 150nm, or from about 60 nm to about 120 nm, respectively.

In another preferred embodiment of the invention the lipid nanoparticleshave a hydrodynamic diameter in the range from about 50 nm to about 300nm, or from about 60 nm to about 250 nm, from about 60 nm to about 150nm, or from about 60 nm to about 120 nm, respectively.

In embodiments where more than one or a plurality, e.g. 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15 of nucleic acid species of the inventionare comprised in the composition, said more than one or said pluralitye.g. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 of nucleic acidspecies of the invention may be complexed within one or more lipidsthereby forming LNPs comprising more than one or a plurality, e.g. 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 of different nucleic acidspecies.

According to a preferred embodiment the lipid-based carriers preferablyencapsulating or comprising RNA are purified by at least onepurification step, preferably by at least one step of TFF and/or atleast one step of clarification and/or at least one step of filtration.This purification particularly leads to reducing the amount of ethanolin the composition, which has been used for the lipid formulation.

In this context it is particularly preferred that the compositioncomprises after purification less than about 500 ppM ethanol, preferablyless than about 50 ppM ethanol, more preferably less than about 5 ppMethanol.

In embodiments, the LNPs described herein may be lyophilized in order toimprove storage stability of the formulation and/or the nucleic acid,preferably the RNA. In embodiments, the LNPs described herein may bespray dried in order to improve storage stability of the formulationand/or the nucleic acid. Lyoprotectants for lyophilization and or spraydrying may be selected from trehalose, sucrose, mannose, dextran andinulin. A preferred lyoprotectant is sucrose, optionally comprising afurther lyoprotectant. A further preferred lyoprotectant is trehalose,optionally comprising a further lyoprotectant.

Accordingly, the composition, e.g. the composition comprising LNPs islyophilized (e.g. according to WO2016/165831 or WO2011/069586) to yielda temperature stable dried nucleic acid (powder) composition as definedherein (e.g. RNA or DNA). The composition, e.g. the compositioncomprising LNPs may also be dried using spray-drying or spray-freezedrying (e.g. according to WO2016/184575 or WO2016/184576) to yield atemperature stable composition (powder) as defined herein.

Accordingly, in preferred embodiments, the composition is a driedcomposition.

The term “dried composition” as used herein has to be understood ascomposition that has been lyophilized, or spray-dried, or spray-freezedried as defined above to obtain a temperature stable dried composition(powder) e.g. comprising LNP complexed RNA (as defined above).

According to further embodiments, the composition of the second aspectmay comprise at least one adjuvant.

Suitably, the adjuvant is preferably added to enhance theimmunostimulatory properties of the composition.

The term “adjuvant” as used herein will be recognized and understood bythe person of ordinary skill in the art, and is for example intended torefer to a pharmacological and/or immunological agent that may modify,e.g. enhance, the effect of other agents or that may be suitable tosupport administration and delivery of the composition. The term“adjuvant” refers to a broad spectrum of substances. Typically, thesesubstances are able to increase the immunogenicity of antigens. Forexample, adjuvants may be recognized by the innate immune systems and,e.g., may elicit an innate immune response (that is, a non-specificimmune response). “Adjuvants” typically do not elicit an adaptive immuneresponse. In the context of the invention, adjuvants may enhance theeffect of the antigenic peptide or protein provided by the nucleic acid.In that context, the at least one adjuvant may be selected from anyadjuvant known to a skilled person and suitable for the present case,i.e. supporting the induction of an immune response in a subject, e.g.in a human subject.

Accordingly, the composition of the second aspect may comprise at leastone adjuvant, wherein the at least one adjuvant may be suitably selectedfrom any adjuvant provided in WO2016/203025. Adjuvants disclosed in anyof the claims 2 to 17 of WO2016/203025, preferably adjuvants disclosedin claim 17 of WO2016/203025 are particularly suitable, the specificcontent relating thereto herewith incorporated by reference. Adjuvantsmay suitably used and comprised in the composition of the second aspect,or the vaccine of the forth aspect, to e.g. reduce the amount of nucleicacid required for a sufficient immune response against the encodedprotein and/or to improve the efficacy of the composition/the vaccinefor treatment/vaccination of the elderly. A suitable adjuvant in thecontext of a coronavirus composition or vaccine (in particular forcompositions comprising a polypeptide of the third aspect) may be aToll-like receptor 9 (TLR9) agonist adjuvant, CpG 1018TM.

The composition of the second aspect may comprise, besides thecomponents specified herein, at least one further component which may beselected from the group consisting of further antigens (e.g. in the formof a peptide or protein, preferably derived from a coronavirus) orfurther antigen-encoding nucleic acids (preferably encoding peptide orprotein, preferably derived from a coronavirus); a furtherimmunotherapeutic agent; one or more auxiliary substances (cytokines,such as monokines, lymphokines, interleukins or chemokines); or anyfurther compound, which is known to be immune stimulating due to itsbinding affinity (as ligands) to human Toll-like receptors; and/or anadjuvant nucleic acid, preferably an immunostimulatory RNA (isRNA), e.g.CpG-RNA etc.

In embodiments, a composition comprising lipid-based carriersencapsulating an RNA is stable after storage as a liquid, for examplestable for at least 2 weeks after storage as a liquid at temperatures ofabout 5° C.

In some aspects, as used herein, “stable” refers to a liquid compositioncomprising lipid-based carriers encapsulating an RNA where the measuredvalues for various physiochemical parameters are within a defined rangeafter storage. In one embodiment, the liquid composition comprisinglipid-based carriers encapsulating an RNA is analyzed to assessstability according to various parameters. Suitable stability parametersinclude, without limitation, RNA integrity, Z-average particle size,polydispersity index (PDI), the amount of free RNA in the liquidcomposition, encapsulation efficiency of the RNA (proportion of the RNAin percent incorporated with lipid-based carriers), shape and morphologyof the lipid-based carriers encapsulating an RNA, pH, osmolality, orturbidity. Further, “stable” refers to a liquid composition comprisinglipid-based carriers encapsulating an RNA where the measured values forvarious functional parameters are within a defined range after storage.In one embodiment, the liquid composition comprising lipid-basedcarriers encapsulating an RNA is analyzed to assess the potency of theliquid composition including for example the expression of the encodedpeptide or protein, the induction of specific antibody titers, theinduction of neutralizing antibody titers, the induction of T-cell, thereactogenicity of the liquid composition including for example theinduction of innate immune responses ect.

In preferred embodiments, the term “stable” refers to RNA integrity.

In various embodiments, a composition of the embodiments is defined as atemperature stable liquid pharmaceutical composition and comprises acertain concentration of lipid-based carriers encapsulating an RNA whichmay be a suitable feature for achieving a temperature stability of theliquid composition. Without whishing to be bound to theory, a certainconcentration of the RNA may have advantageous effects on temperaturestability of the composition when stored as a liquid.

In embodiments, the concentration of the RNA in a composition is in arange of about 10 μg/ml to about 10 mg/ml. In embodiments, theconcentration of the RNA in a liquid composition is in a range of about100 μg/ml to about 5 mg/ml. In embodiments, the concentration of the RNAin the liquid composition is in a range of about 100 μg/ml to about 2mg/ml. In embodiments, the concentration of the RNA in a liquidcomposition is in a range of about 100 μg/ml to about 1 mg/ml. Inembodiments, the concentration of the RNA in a liquid composition is ina range of about 200 μg/ml to about 1 mg/ml. In embodiments, theconcentration of RNA in a liquid composition is in a range of about 100μg/ml to about 500 μg/ml. In preferred embodiments, the concentration ofRNA in a liquid composition is in a range of about 200 μg/ml to about500 μg/ml. In preferred embodiments, the concentration of RNA in theliquid composition is in a range of about 200 μg/ml to about 600 μg/ml.In preferred embodiments, the concentration of RNA in the liquidcomposition is in a range of about 200 μg/ml to about 700 μg/ml. Inpreferred embodiments, the concentration of RNA in the liquidcomposition is in a range of about 200 μg/ml to about 800 μg/ml. Inpreferred embodiments, the concentration of RNA in the liquidcomposition is in a range of about 200 μg/ml to about 900 μg/ml.

In embodiments, the concentration of RNA in a composition is for exampleabout 100 μg/ml, about 200 μg/ml, about 300 μg/ml, about 400 μg/ml,about 500 μg/ml, about 600 μg/ml, about 700 μg/ml, about 800 μg/ml,about 900 μg/ml, about 1 mg/ml. In preferred embodiments, theconcentration of the RNA in the liquid composition is at least 100μg/ml, preferably at least 200 μg/ml, more preferably at least 500μg/ml.

In various embodiments, RNA of a pharmaceutical composition has acertain RNA integrity which may be a suitable feature for achieving atemperature stability of the liquid composition.

The term “RNA integrity” generally describes whether the complete RNAsequence is present in the liquid composition. Low RNA integrity couldbe due to, amongst others, RNA degradation, RNA cleavage, incorrect orincomplete chemical synthesis of the RNA, incorrect base pairing,integration of modified nucleotides or the modification of alreadyintegrated nucleotides, lack of capping or incomplete capping, lack ofpolyadenylation or incomplete polyadenylation, or incomplete RNA invitro transcription. RNA is a fragile molecule that can easily degrade,which may be caused e.g. by temperature, ribonucleases, pH or otherfactors (e.g. nucleophilic attacks, hydrolysis etc.), which may reducethe RNA integrity and, consequently, the functionality of the RNA.

The skilled person can choose from a variety of differentchromatographic or electrophoretic methods for determining an RNAintegrity. Chromatographic and electrophoretic methods are well-known inthe art. In case chromatography is used (e.g. RP-HPLC), the analysis ofthe integrity of the RNA may be based on determining the peak area (or“area under the peak”) of the full length RNA in a correspondingchromatogram. The peak area may be determined by any suitable softwarewhich evaluates the signals of the detector system. The process ofdetermining the peak area is also referred to as integration. The peakarea representing the full length RNA is typically set in relation tothe peak area of the total RNA in a respective sample. The RNA integritymay be expressed in % RNA integrity.

In the context of aspects of the invention, RNA integrity may bedetermined using analytical (RP)HPLC. Typically, a test sample of theliquid composition comprising lipid based carrier encapsulating RNA maybe treated with a detergent (e.g. about 2% Triton X100) to dissociatethe lipid based carrier and to release the encapsulated RNA. Thereleased RNA may be captured using suitable binding compounds, e.g.Agencourt AMPure XP beads (Beckman Coulter, Brea, Calif., USA)essentially according to the manufacturer's instructions. Followingpreparation of the RNA sample, analytical (RP)HPLC may be performed todetermine the integrity of RNA. Typically, for determining RNAintegrity, the RNA samples may be diluted to a concentration of 0.1 g/lusing e.g. water for injection (WFI). About 10 μl of the diluted RNAsample may be injected into an HPLC column (e.g. a monolithicpoly(styrene-divinylbenzene) matrix). Analytical (RP)HPLC may beperformed using standard conditions, for example: Gradient 1: Buffer A(0.1M TEAA (pH 7.0)); Buffer B (0.1M TEAA (pH 7.0) containing 25%acetonitrile). Starting at 30% buffer B the gradient extended to 32%buffer B in 2 min, followed by an extension to 55% buffer B over 15minutes at a flow rate of 1 ml/min. HPLC chromatograms are typicallyrecorded at a wavelength of 260 nm. The obtained chromatograms may beevaluated using a software and the relative peak area may be determinedin percent (%) as commonly known in the art. The relative peak areaindicates the amount of RNA that has 100% RNA integrity. Since theamount of the RNA injected into the HPLC is typically known, theanalysis of the relative peak area provides information on the integrityof the RNA. Thus, if e.g. 100 ng RNA have been injected in total, and100 ng are determined as the relative peak area, the RNA integrity wouldbe 100%. If, for example, the relative peak area would correspond to 80ng, the RNA integrity would be 80%. Accordingly, RNA integrity in thecontext of the invention is determined using analytical HPLC, preferablyanalytical RP-HPLC.

In certain embodiments, a pharmaceutical of the embodiments is stablefor about 2 weeks to about 1 month, 2 months, 3 months, 4 months, 5months, 6 months or 1 year after storage as a liquid at temperatures ofabout 5° C. For example, at least about 50%, 60%, 70%, 75%, 80%, 85%,90% or 95% of the RNA remains intact after storage as a liquid for atleast about 5° C. for about two weeks, three weeks, one month, 6 weeks,2 months, 3 months, 4 months, 5 months, 6 months or 1 year. In someaspects, a temperature stable liquid pharmaceutical of the embodimentscomprises at least about 70%, 75%, 80%, 85%, 90% or 95% of intact RNA atleast about two weeks after storage as a liquid at temperatures of about5° C. In further aspects, a temperature stable liquid pharmaceutical ofthe embodiments comprises at least about 70%, 75%, 80%, 85%, 90% or 95%of intact RNA at least 1 month after storage as a liquid at temperaturesof about 5° C. In certain aspects, a temperature stable liquidpharmaceutical of the embodiments comprises at least about 70%, 75%,80%, 85%, 90% or 95% of intact RNA at least about 2 weeks to about 1month, 2 months, 3 months, 4 months, 5 months, 6 months or 1 year afterstorage as a liquid at temperatures of about 5° C. In some specificaspects, a temperature stable liquid pharmaceutical of the embodimentscomprises at least about 80% of intact RNA after about two weeks ofstorage as a liquid at temperatures of about 5° C.

In certain embodiments, RNA of a composition has an RNA integrityranging from about 40% to about 100%. In embodiments, the RNA has an RNAintegrity ranging from about 50% to about 100%. In embodiments, the RNAhas an RNA integrity ranging from about 60% to about 100%. Inembodiments, the RNA has an RNA integrity ranging from about 70% toabout 100%. In embodiments, the RNA integrity is for example about 50%,about 60%, about 70%, about 80%, or about 90%. RNA is suitablydetermined using analytical HPLC, preferably analytical RP-HPLC.

In preferred embodiments, the RNA of a composition has an RNA integrityof at least about 50%, preferably of at least about 60%, more preferablyof at least about 70%, most preferably of at least about 80% or ABOUT90%. RNA is suitably determined using analytical HPLC, preferablyanalytical RP-HPLC.

In various embodiments, nucleic acid, e.g., RNA of a pharmaceuticalcomposition does not exceed a certain proportion of free RNA, which maybe a suitable feature for achieving a temperature stability of theliquid composition. Without wishing to be bound to theory, free RNA inthe liquid composition may be more vulnerable to degradation as the RNAthat is encapsulated in the lipid based carrier.

In certain aspects, compositions of the embodiments comprise RNA. Inthis context, the term “free RNA” or “non-complexed RNA” or“non-encapsulated RNA” comprise the RNA molecules that are notencapsulated in the lipid-based carriers as defined herein. Duringformulation of the liquid composition (e.g. during encapsulation of theRNA into the lipid-based carriers), free RNA may represent acontamination or an impurity. A large proportion of non-encapsulated orfree RNA may also be an indicator for destabilization of a lipid-basedcarriers of the composition (e.g. upon storage of the composition). Forexample, free RNA detectable in the liquid composition may increaseduring storage, which may be used as a feature to determine thetemperature stability of the composition.

The skilled person can choose from a variety of different methods fordetermining the amount and/or the proportion of free nucleic acid offree RNA in the liquid composition. Free RNA in the liquid compositionmay be determined by chromatographic methods (e.g. AEX, SEC) or by usingprobes (e.g. dyes) that bind to free RNA in the composition. In thecontext of the invention, the amount of free RNA or non-encapsulated RNAmay be determined using a dye based assay. Suitable dyes that may beused to determine the amount and/or the proportion of free RNA compriseRiboGreen®, PicoGreen® dye, OilGreen® dye, QuantiFluor® RNA dye, Qubit®RNA dye, Quant-iT™ RNA dye, TOTO®-1 dye, YOYO®-1 dye. Such dyes aresuitable to discriminate between free RNA and encapsulated RNA.Reference standards consisting of defined amounts of free RNA orencapsulated RNA may be used and mixed with the respective reagent (e.g.RiboGreen® reagent (Excitation 500 nm/Emission 525 nm)) as recommendedby the supplier's instructions. Typically, the free RNA of the liquidcomposition is quantitated using the Quant-iT RiboGreen RNA Reagentaccording to the manufacturer's instructions. The proportion of free RNAin the context of the invention is typically determined using aRiboGreen assay.

In embodiments, a composition comprises free nucleic acid, such as freeRNA ranging from about 30% to about 0%. In embodiments, the liquidcomposition comprises about 20% free RNA (and about 80% encapsulatedRNA), about 15% free RNA (and about 85% encapsulated RNA), about 10%free RNA (and about 90% encapsulated RNA), or about 5% free RNA (andabout 95% encapsulated RNA). In preferred embodiments, the liquidcomposition comprises less than about 20% free RNA, preferably less thanabout 15% free RNA, more preferably less than about 10% free RNA, mostpreferably less than about 5% free RNA.

In aspects comprising RNA nucleic acids, the term “encapsulated RNA”comprise the RNA molecules that are encapsulated in the lipid-basedcarriers as defined herein. The proportion of encapsulated RNA in thecontext of the invention is typically determined using a RiboGreenassay.

Accordingly, in embodiments, about 70% to about 100% of the RNA in theliquid composition is encapsulated in the lipid-based carriers. Inembodiments, the liquid composition comprises about 80% encapsulated RNA(and about 20% free RNA), about 85% encapsulated RNA (and about 15% freeRNA), about 90% encapsulated RNA (and about 10% free RNA), or about 95%encapsulated RNA (and 5% about free RNA).

In preferred embodiments, 80% of the nucleic acid (e.g., RNA) comprisedin the liquid composition is encapsulated, preferably 85% of the RNAcomprised in the composition is encapsulated, more preferably 90% of theRNA comprised in the composition is encapsulated, most preferably 95% ofthe RNA comprised in the composition is encapsulated.

In various embodiments, a pharmaceutical composition (in particular theRNA of the composition) does not exceed a certain amount of divalentcations, which may be a suitable feature for achieving temperaturestability of the liquid composition. Divalent cations, e.g. divalentmetal ions (e.g. Mg2+, Ca2+, Mn2+, Zn2+, Ni2+, Fe2+, Co2+, Pb2+) maycause hydrolysis of the RNA encapsulated in the lipid-based carriersduring storage as a liquid.

In some aspects, RNA of a composition may typically be produced by RNAin vitro transcription (IVT) of a (linear) DNA template. Common RNA invitro transcription buffers comprise large amounts of MgCl2 (e.g. 5 mM,15 mM or more) which is a co-factor of the RNA polymerase. Accordingly,the obtained in vitro transcribed RNA may comprise Mg2+ ions as acontamination. After RNA in vitro transcription, the DNA template istypically removed by means of DNAses. Common buffers for DNAse digestcomprise large amounts of CaCl₂) (e.g. 1 mM, 5 mM or more) which is aco-factor of the DNAse. Accordingly, the obtained in vitro transcribedRNA may comprise Ca2+ as a contamination.

Typically, various RNA purification steps (e.g. RP-HPLC, tangential flowfiltration) may be employed to remove various contaminations includingdivalent metal ions. Suitably, the RNA used for encapsulation in thelipid-based carriers of the invention has been purified to removedivalent metal ions.

In embodiments, a composition of the embodiments comprises less thanabout 100 nM divalent cations per g RNA, preferably less than about 50nM divalent cations per g RNA, more preferably less than about 10 nMdivalent cations per g RNA. In embodiments, the divalent cations areselected from Mg2+ and/or Ca2+. In embodiments, the liquid compositioncomprises less than about 100 nM Mg2+ per g RNA. In embodiments, theliquid composition comprises less than about 100 nM Ca2 per g RNA.Typically, Ion Chromatography (IC) coupled with Inductively CoupledPlasma Mass Spectrometry (IC-ICP-MS) may be used for determination ofdivalent cations.

In embodiments, the lipid-based carrier encapsulating the RNA of acomposition comprises less than about 100 nM divalent cations per g RNA,preferably less than about 50 nM divalent cations per g RNA, morepreferably less than about 10 nM divalent cations per g RNA. Inembodiments, the divalent cations are selected from Mg2+ and/or Ca2+. Inembodiments, the lipid-based carriers encapsulating the RNA compriseless than about 100 nM Mg2+ per g RNA. In embodiments, the lipid-basedcarriers encapsulating the RNA comprises less than about 100 nM Ca2 perg RNA. Typically, Ion Chromatography (IC) coupled with InductivelyCoupled Plasma Mass Spectrometry (IC-ICP-MS) may be used fordetermination of Mg2+ and/or Ca2+.

In embodiments, the RNA of a composition comprises Na+ as a counter ion.In embodiments, the RNA comprises Na+ in an amount ranging from about 10μg Na+ per g RNA to about 1 mg Na+ per g RNA. In embodiments, the RNAcomprises Na+ as a counter ion in an amount of at least about 100 μg Na+per g RNA, preferably at least about 200 μg Na+ per g RNA. Typically,Ion Chromatography (IC) coupled with Inductively Coupled Plasma MassSpectrometry (IC-ICP-MS) may be used for determination of Na+.

In embodiments, the composition comprises at least one antagonist of atleast one RNA sensing pattern recognition receptor. Such an antagonistmay preferably be co-formulated in lipid-based carriers as definedherein.

Suitable antagonist of at least one RNA sensing pattern recognitionreceptor are disclosed in PCT patent application PCT/EP2020/072516, thefull disclosure herewith incorporated by reference. In particular, thedisclosure relating to suitable antagonist of at least one RNA sensingpattern recognition receptors as defined in any one of the claims 1 to94 of PCT/EP2020/072516 are incorporated.

In preferred embodiments, the composition comprises at least oneantagonist of at least one RNA sensing pattern recognition receptorselected from a Toll-like receptor, preferably TLR7 and/or TLR8.

In embodiments, the at least one antagonist of at least one RNA sensingpattern recognition receptor is selected from a nucleotide, a nucleotideanalog, a nucleic acid, a peptide, a protein, a small molecule, a lipid,or a fragment, variant or derivative of any of these.

In preferred embodiments, the at least one antagonist of at least oneRNA sensing pattern recognition receptor is a single strandedoligonucleotide, preferably a single stranded RNA Oligonucleotide.

In embodiments, the antagonist of at least one RNA sensing patternrecognition receptor is a single stranded oligonucleotide that comprisesor consists of a nucleic acid sequence identical or at least 70%, 80%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identical to a nucleic acid sequence selected from the groupconsisting of SEQ ID NOs: 85-212 of PCT/EP2020/072516, or fragments ofany of these sequences.

In preferred embodiments, the antagonist of at least one RNA sensingpattern recognition receptor is a single stranded oligonucleotide thatcomprises or consists of a nucleic acid sequence identical or at least70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% identical to a nucleic acid sequence selected from thegroup consisting of SEQ ID NOs: 85-87, 149-212 of PCT/EP2020/072516, orfragments of any of these sequences.

A particularly preferred antagonist of at least one RNA sensing patternrecognition receptor in the context of the invention is 5′-GAG CGmGCCA-3′ (SEQ ID NO: 85 of PCT/EP2020/072516), or a fragment thereof.

In embodiments, the molar ratio of the at least one antagonist of atleast one RNA sensing pattern recognition receptor as defined herein tothe at least one nucleic acid, preferably RNA encoding a SRAS-CoV-2antigenic peptide or protein as defined herein suitably ranges fromabout 1:1, to about 100:1, or ranges from about 20:1, to about 80:1.

In embodiments, the wherein the weight to weight ratio of the at leastone antagonist of at least one RNA sensing pattern recognition receptoras defined herein to the at least one nucleic acid, preferably RNAencoding a SRAS-CoV-2 antigenic peptide or protein as defined hereinsuitably ranges from about 1:1, to about 1:30, or ranges from about 1:2,to about 1:10.

Polypeptide for a Coronavirus Vaccine

In a third aspect, the present invention provides an antigenicpolypeptide suitable for a coronavirus vaccine, in particular for aSARS-CoV-2 (formerly nCoV-2019) coronavirus vaccine. In preferredembodiments, the polypeptide is derived from any protein or fragmentthereof that a nucleic acid of the first aspect is encoding. Preferredpolypeptide designs are disclosed in List 1.

In preferred embodiments, the amino acid sequences of the antigenicpolypeptide of the third aspect is identical or at least 70%, 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to any one of amino acid sequences SEQ ID NOs: 1-111,274-11663, 13176-13510, 13521-14123, 22732-22758, 22917, 22923,22929-22964, 26938, 26939 or fragment or variant of any of these.

The polypeptide of the third aspect may also be comprised in a(pharmaceutical) composition, including pharmaceutically acceptablecarriers, or adjuvants as defined herein, in particular as defined inthe context of the second aspect. Accordingly, the invention alsorelates to a (pharmaceutical) composition comprising said antigenicpolypeptide.

The polypeptide of the third aspect may also be comprised in vaccine,including pharmaceutically acceptable carriers, or adjuvants as definedherein, in particular as defined in the context of the fourth aspect.Accordingly, the invention also relates to a vaccine comprising saidantigenic polypeptide (see fourth aspect). Suitable adjuvants that maybe used in combination with a polypeptide as defined herein aresaponin-based adjuvants (steroid or triterpenoid glycosides), e.g.Matrix-M adjuvant.

Vaccine:

In a fourth aspect, the present invention provides a vaccine against acoronavirus, preferably against a SARS-CoV-2 (formerly nCoV-2019)coronavirus causing COVID-19 disease.

In preferred embodiments of the fourth aspect, the vaccine comprises atleast one nucleic acid (e.g. DNA or RNA), preferably at least one RNA ofthe first aspect, or the composition of the second aspect.

In other embodiments, the vaccine comprises at least one polypeptide asdefined in the third aspect.

In other embodiments, the vaccine comprises at least one plasmid DNA oradenovirus DNA as defined in the first aspect.

Notably, embodiments relating to the composition of the second aspectmay likewise be read on and be understood as suitable embodiments of thevaccine of the fourth aspect. Also, embodiments relating to the vaccineof the fourth aspect may likewise be read on and be understood assuitable embodiments of the composition of the second aspect.Furthermore, features and embodiments described in the context of thefirst aspect (the nucleic acid of the invention) have to be read on andhave to be understood as suitable embodiments of the composition of thefourth aspect.

The term “vaccine” will be recognized and understood by the person ofordinary skill in the art, and is for example intended to be aprophylactic or therapeutic material providing at least one epitope orantigen, preferably an immunogen. In the context of the invention theantigen or antigenic function is suitably provided by the inventivenucleic acid of the first aspect (said nucleic acid comprising a codingsequence encoding a antigenic peptide or protein derived from aSARS-CoV-2 coronavirus) or the composition of the second aspect(comprising at least one nucleic acid of the first aspect). In otherembodiments, the antigen or antigenic function is provided by theinventive polypeptide of the third aspect.

In preferred embodiments, the vaccine, or the composition of the secondaspect, elicits an adaptive immune response, preferably an adaptiveimmune response against a coronavirus, preferably against SARS-CoV-2coronavirus.

In particularly preferred embodiments, the vaccine, or the compositionof the second aspect, elicits functional antibodies that can effectivelyneutralize the virus, preferably SARS-CoV-2 coronavirus.

In further preferred embodiments, the vaccine, or the composition of thesecond aspect, elicits mucosal IgA immunity by inducing of mucosal IgAantibodies.

In particularly preferred embodiments, the vaccine, or the compositionof the second aspect, elicits functional antibodies that can effectivelyneutralize the virus, preferably SARS-CoV-2 coronavirus.

In further particularly preferred embodiments, the vaccine, or thecomposition of the second aspect, induces broad, functional cellularT-cell responses against coronavirus, preferably against SARS-CoV-2coronavirus.

In further particularly preferred embodiments, the vaccine, or thecomposition of the second aspect, induces a well-balanced B cell and Tcell response against coronavirus, preferably against SARS-CoV-2coronavirus.

According to a preferred embodiment, the vaccine as defined herein mayfurther comprise a pharmaceutically acceptable carrier and optionally atleast one adjuvant as specified in the context of the second aspect.

Suitable adjuvants in that context may be selected from adjuvantsdisclosed in claim 17 of WO2016/203025.

In a preferred embodiment, the vaccine is a monovalent vaccine.

The terms “monovalent vaccine”, “monovalent composition” “univalentvaccine” or “univalent composition” will be recognized and understood bythe person of ordinary skill in the art, and are e.g. intended to referto a composition or a vaccine comprising only one antigen or antigenconstruct from a pathogen. Accordingly, said vaccine or compositioncomprises only one nucleic acid species encoding a single antigen orantigen construct of a single organism. The term “monovalent vaccine”includes the immunization against a single valence. In the context ofthe invention, a monovalent SARS-CoV-2 coronavirus vaccine orcomposition would comprise at least one nucleic acid encoding one singleantigenic peptide or protein derived from one specific SARS-CoV-2coronavirus.

In embodiments, the vaccine is a polyvalent vaccine comprising aplurality or at least more than one of the nucleic acid species definedin the context of the first aspect. Embodiments relating to a polyvalentcomposition as disclosed in the context of the second aspect maylikewise be read on and be understood as suitable embodiments of thepolyvalent vaccine.

The terms “polyvalent vaccine”, “polyvalent composition” “multivalentvaccine” or “multivalent composition” will be recognized and understoodby the person of ordinary skill in the art, and are e.g. intended torefer to a composition or a vaccine comprising antigens from more thanone virus (e.g. different SARS-CoV-2 coronavirus isolates), orcomprising different antigens or antigen constructs of the sameSARS-CoV-2 coronavirus, or any combination thereof. The terms describethat said vaccine or composition has more than one valence. In thecontext of the invention, a polyvalent SARS-CoV-2 coronavirus vaccinewould comprise nucleic acid sequences encoding antigenic peptides orproteins derived from several different SARS-CoV-2 coronavirus (e.g.different SARS-CoV-2 coronavirus isolates) or comprising nucleic acidsequences encoding different antigens or antigen constructs from thesame SARS-CoV-2 coronavirus, or a combination thereof.

In preferred embodiments, the polyvalent or multivalent vaccinecomprises at least one polyvalent composition as defined in the secondaspect. Particularly preferred are polyvalent compositions as defined insection “Multivalent compositions of the invention”.

In embodiments, the vaccine comprises at least one antagonist of atleast one RNA sensing pattern recognition receptor as defined in thesecond aspect.

The coronavirus vaccine typically comprises a safe and effective amountof nucleic acid (e.g. DNA or RNA), preferably RNA of the first aspect orcomposition of the second aspect (or the polypeptide of the thirdaspect). As used herein, “safe and effective amount” means an amount ofnucleic acid or composition sufficient to significantly induce apositive modification of a disease or disorder related to an infectionwith coronavirus, preferably SARS-CoV-2 coronavirus. At the same time, a“safe and effective amount” is small enough to avoid seriousside-effects. In relation to the nucleic acid, composition, or vaccineof the present invention, the expression “safe and effective amount”preferably means an amount of nucleic acid, composition, or vaccine thatis suitable for stimulating the adaptive immune system againstcoronavirus in such a manner that no excessive or damaging immunereactions (e.g. innate immune responses) are achieved.

A “safe and effective amount” of the nucleic acid, composition, orvaccine as defined above will vary in connection with the particularcondition to be treated and also with the age and physical condition ofthe patient to be treated, the severity of the condition, the durationof the treatment, the nature of the accompanying therapy, of theparticular pharmaceutically acceptable carrier used, and similarfactors, within the knowledge and experience of the skilled person.Moreover, the “safe and effective amount” of the nucleic acid, thecomposition, or vaccine may depend from application/delivery route(intradermal, intramuscular, intranasal), application device (jetinjection, needle injection, microneedle patch, electroporation device)and/or complexation/formulation (protamine complexation or LNPencapsulation, DNA or RNA). Moreover, the “safe and effective amount” ofthe nucleic acid, the composition, or the vaccine may depend from thephysical condition of the treated subject (infant, pregnant women,immunocompromised human subject etc.).

The coronavirus vaccine can be used according to the invention for humanmedical purposes and also for veterinary medical purposes (mammals,vertebrates, or avian species).

The pharmaceutically acceptable carrier as used herein preferablyincludes the liquid or non-liquid basis of the inventive coronavirusvaccine. If the inventive vaccine is provided in liquid form, thecarrier will be water, typically pyrogen-free water; isotonic saline orbuffered (aqueous) solutions, e.g. phosphate, citrate etc. bufferedsolutions. Preferably, Ringer-Lactate solution is used as a liquid basisfor the vaccine or the composition according to the invention asdescribed in WO2006/122828, the disclosure relating to suitable bufferedsolutions incorporated herewith by reference. Other preferred solutionsused as a liquid basis for the vaccine or the composition, in particularfor compositions/vaccines comprising LNPs, comprise sucrose and/ortrehalose.

The choice of a pharmaceutically acceptable carrier as defined herein isdetermined, in principle, by the manner, in which the pharmaceuticalcomposition(s) or vaccine according to the invention is administered.The coronavirus vaccine is preferably administered locally. Routes forlocal administration in general include, for example, topicaladministration routes but also intradermal, transdermal, subcutaneous,or intramuscular injections or intralesional, intracranial,intrapulmonal, intracardial, intraarticular and sublingual injections.More preferably, composition or vaccines according to the presentinvention may be administered by an intradermal, subcutaneous, orintramuscular route, preferably by injection, which may be needle-freeand/or needle injection. Preferred in the context of the invention isintramuscular injection. Compositions/vaccines are therefore preferablyformulated in liquid or solid form. The suitable amount of the vaccineor composition according to the invention to be administered can bedetermined by routine experiments, e.g. by using animal models. Suchmodels include, without implying any limitation, rabbit, sheep, mouse,rat, dog and non-human primate models. Preferred unit dose forms forinjection include sterile solutions of water, physiological saline ormixtures thereof. The pH of such solutions should be adjusted to about7.4.

The inventive coronavirus vaccine or composition as defined herein maycomprise one or more auxiliary substances or adjuvants as defined abovein order to further increase the immunogenicity. A synergistic action ofthe nucleic acid contained in the inventive composition/vaccine and ofan auxiliary substance, which may be optionally be co-formulated (orseparately formulated) with the inventive vaccine or composition asdescribed above, is preferably achieved thereby. Such immunogenicityincreasing agents or compounds may be provided separately (notco-formulated with the inventive vaccine or composition) andadministered individually.

The coronavirus vaccine is preferably provided in lyophilized orspray-dried form (as described in the context of the second aspect).Such a lyophilized or spray-dried vaccine is typically comprisestrehalose and/or sucrose and is re-constituted in a suitable liquidbuffer before administration to a subject. In some aspects, alyophilized vaccine of the embodiments comprises mRNA of the embodimentscomplexed with LNPs. In some aspects, a lyophilized composition has awater content of less than about 10%. For example, a lyophilizedcomposition can have a water content of about 0.1% to 10%, 0.1% to 7.5%,or 0.5% to 7.5%, preferably the lyophilized composition has a watercontent of about 0.5% to about 5.0%.

In preferred embodiments administration of a therapeutically effectiveamount of the nucleic acid, the composition, the polypeptide, thevaccine to a subject induces a neutralizing antibody titer againstSARS-CoV-2 coronavirus in the subject.

In some embodiments, the neutralizing antibody titer is at least 100neutralizing units per milliliter (NU/mL), at least 500 NU/mL, or atleast 1000 NU/mL.

In some embodiments, detectable levels of the coronavirus antigen areproduced in the subject at about 1 to about 72 hours post administrationof the nucleic acid, the composition, the polypeptide, or the vaccine.

In some embodiments, a neutralizing antibody titer (against coronavirus)of at least 100 NU/ml, at least 500 NU/ml, or at least 1000 NU/ml isproduced in the serum of the subject at about 1 day to about 72 dayspost administration of the nucleic acid, the composition, thepolypeptide, or the vaccine.

In some embodiments, the neutralizing antibody titer is sufficient toreduce coronavirus infection by at least 50% relative to a neutralizingantibody titer of an unvaccinated control subject or relative to aneutralizing antibody titer of a subject vaccinated with a liveattenuated viral vaccine, an inactivated viral vaccine, or a protein subunit viral vaccine.

In some embodiments, the neutralizing antibody titer and/or a T cellimmune response is sufficient to reduce the rate of asymptomatic viralinfection relative to the neutralizing antibody titer of unvaccinatedcontrol subjects.

In some embodiments, the neutralizing antibody titer and/or a T cellimmune response is sufficient to prevent viral latency in the subject.

In some embodiments, the neutralizing antibody titer is sufficient toblock fusion of virus with epithelial cells of the subject.

In some embodiments, the neutralizing antibody titer is induced within20 days following a single 1 ug-100 ug dose of the nucleic acid, thecomposition, the polypeptide, or the vaccine, or within 40 daysfollowing a second 1 ug-100 μg dose of the nucleic acid, thecomposition, the polypeptide, or the vaccine.

In preferred embodiments, administration of a therapeutically effectiveamount of the nucleic acid, the composition, the polypeptide, or thevaccine to a subject induces a T cell immune response againstcoronavirus in the subject. In preferred embodiments, the T cell immuneresponse comprises a CD4+ T cell immune response and/or a CD8+ T cellimmune response.

Kit or Kit of Parts, Application, Medical Uses, Method of Treatment:

In a fifth aspect, the present invention provides a kit or kit of partssuitable for treating or preventing a coronavirus infection. Preferably,said kit or kit of parts is suitable for treating or preventing acoronavirus, preferably a SARS-CoV-2 (formerly nCoV-2019) coronavirusinfection.

Notably, embodiments relating to the nucleic acid of the first aspect,the composition of the second aspect, the polypeptide of the thirdaspect, and the vaccine of the fourth aspect may likewise be read on andbe understood as suitable embodiments of the kit or kit of parts of thefifth aspect of the invention.

In preferred embodiments, the kit or kit of parts comprises at least onenucleic acid (e.g. RNA or DNA), preferably at least one RNA of the firstaspect, at least one composition of the second aspect, and/or at leastone polypeptide of the third aspect, and/or at least one vaccine of theforth aspect.

In embodiments, the kit or kit of parts comprises at least one DNA asdefined in the first aspect, e.g. at least one plasmid DNA and/or atleast one adenovirus DNA.

In embodiments, the kit or kit of parts comprises at least onepolypeptide as defined in the third aspect.

In addition, the kit or kit of parts may comprise a liquid vehicle forsolubilising, and/or technical instructions providing information onadministration and dosage of the components.

The kit may further comprise additional components as described in thecontext of the composition of the second aspect, and/or the vaccine ofthe forth aspect.

The technical instructions of said kit may contain information aboutadministration and dosage and patient groups. Such kits, preferably kitsof parts, may be applied e.g. for any of the applications or usesmentioned herein, preferably for the use of the nucleic acid of thefirst aspect, the composition of the second aspect, the polypeptide ofthe third aspect, or the vaccine of the forth aspect, for the treatmentor prophylaxis of an infection or diseases caused by a coronavirus,preferably SARS-CoV-2 coronavirus, or disorders related thereto.

Preferably, the nucleic acid, the composition, the polypeptide, or thevaccine is provided in a separate part of the kit, wherein the nucleicacid, the composition, the polypeptide, or the vaccine is preferablylyophilised.

The kit may further contain as a part a vehicle (e.g. buffer solution)for solubilising the nucleic acid, the composition, the polypeptide, orthe vaccine.

In preferred embodiments, the kit or kit of parts as defined hereincomprises Ringer lactate solution.

In preferred embodiments, the kit or kit of parts as defined hereincomprises a multidose container for administration of thecomposition/the vaccine.

Any of the above kits may be used in a treatment or prophylaxis asdefined herein. More preferably, any of the above kits may be used as avaccine, preferably a vaccine against infections caused by acoronavirus, preferably caused by SARS-CoV-2 coronavirus.

In preferred embodiments, the kit or kit of parts comprises thefollowing components:

-   a) at least one container or vial comprising a composition or    SARS-CoV-2 vaccine as defined herein, wherein the composition or    SARS-CoV-2 vaccine has a nucleic acid concentration, preferably an    RNA concentration in a range of about 100 μg/ml to about 1 mg/ml,    preferably in a range of about 100 μg/ml to about 500 μg/ml, e.g.    about 270 μg/ml.-   b) at least one dilution container or vial comprising a sterile    dilution buffer, suitably a buffer comprising NaCl, optionally    comprising a preservative;-   c) at least one means for transferring the composition or vaccine    from the storage container to the dilution container; and-   d) at least one syringe for administering the final diluted    composition or vaccine to a subject, preferably configured for    intramuscular administration to a human subject, wherein the final    diluted composition or vaccine has a nucleic acid concentration,    preferably an RNA concentration in a range of about 10 μg/ml to    about 100 μg/ml, preferably in a range of about 10 μg/ml to about 50    μg/ml, e.g. about 24 μg/ml

In an embodiment, the kit or kit of parts comprises more than onemRNA-based SARS-CoV-2 composition/vaccine, preferably

-   -   at least one vaccine as defined herein provided in a first vial        or container, wherein the vaccine comprises at least one nucleic        acid, preferably RNA, which is identical or at least 70%, 80%,        85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,        98%, or 99% identical to a nucleic acid sequence of SEQ ID NO.        163, 149 or 24837, preferably formulated in lipid nanoparticles        (LNPs), which have a molar ratio of approximately        50:10:38.5:1.5, preferably 47.5:10:40.8:1.7 or more preferably        47.4:10:40.9:1.7 proportion (mol %) of cationic lipid III-3        (ALC-0315), DSPC, cholesterol and PEG-lipid of formula (IVa)        (with n=49 or with n=45 (ALC-0159)). Preferably, the nucleic        acid, preferably mRNA is not chemically modified.    -   at least one further vaccine as defined herein provided in a        first vial or container, wherein the composition/vaccine        comprises at least one nucleic acid, preferably RNA, which is        identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%,        91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a        nucleic acid sequence of SEQ ID NO: 23311, 23531, 24851, 23310,        23530, 24850, 23313, 23533, 24853, 23314, 23534, or 24854,        preferably formulated in lipid nanoparticles (LNPs), which have        a molar ratio of approximately 50:10:38.5:1.5, preferably        47.5:10:40.8:1.7 or more preferably 47.4:10:40.9:1.7 proportion        (mol %) of cationic lipid III-3 (ALC-0315), DSPC, cholesterol        and PEG-lipid of formula (IVa) (with n=49 or with n=45        (ALC-0159)). Preferably, the nucleic acid, preferably mRNA is        not chemically modified.

In an embodiment, the kit or kit of parts comprises two differentSARS-CoV-2 vaccines for prime vaccination and boost vaccination:

-   -   at least one prime vaccine as defined herein provided in a first        vial or container, wherein the vaccine is an mRNA-based        SARS-CoV-2 vaccine as defined herein; and    -   at least one boost vaccine as defined herein provided in a first        vial or container, wherein the composition/vaccine is an        adenovirus-based SARS-CoV-2 vaccine as defined herein.

In an embodiment, the kit or kit of parts comprises two differentSARS-CoV-2 vaccines for prime vaccination and boost vaccination:

-   -   at least one boost vaccine as defined herein provided in a first        vial or container, wherein the vaccine is an mRNA-based        SARS-CoV-2 vaccine as defined herein; and    -   at least one prime vaccine as defined herein provided in a first        vial or container, wherein the composition/vaccine is an        adenovirus-based SARS-CoV-2 vaccine as defined herein.

Combination:

A sixth aspect relates to a combination of at least two nucleic acidsequences as defined in the first aspect, at least two compositions asdefined in the context of the second aspect, at least two polypeptidesas defined in the third aspect, at least two vaccines as defined in thecontext of the fourth aspect, or at least two kits as defined in thefifth aspect.

In the context of the present invention, the term “combination”preferably means a combined occurrence of at least two components,preferably at least two nucleic acid sequences as defined in the firstaspect, at least two compositions as defined in the context of thesecond aspect, at least two polypeptides as defined in the third aspect,at least two vaccines as defined in the context of the fourth aspect, orat least two kits as defined in the fifth aspect. The components of sucha combination may occur as separate entities. Thus, the administrationof the components of the combination may occur either simultaneously ortimely staggered, either at the same site of administration or atdifferent sites of administration.

Notably, embodiments relating to the nucleic acid of the first aspect,the composition of the second aspect, the polypeptide of the thirdaspect, and the vaccine of the fourth aspect, or the kit or kit of partsof the fifth aspect may likewise be read on and be understood assuitable embodiments of the components of the combination of the sixthaspect.

In embodiments, the combination may comprise a plurality or at leastmore than one of the nucleic acid species, e.g. RNA species as definedin the context of the first aspect of the invention, wherein the nucleicacid species are provided as separate components.

Preferably, the combination as defined herein may comprise 2, 3, 4, 5,6, 7, 8, 9, or 10 different nucleic acids e.g. RNA species as defined inthe context of the first aspect of the invention; 2, 3, 4, 5, 6, 7, 8,9, or 10 different compositions as defined in the context of the secondaspect of the invention; 2, 3, 4, 5, 6, 7, 8, 9, or 10 differentpolypeptides as defined in the context of the third aspect of theinvention; 2, 3, 4, 5, 6, 7, 8, 9, or 10 different vaccines as definedin the context of the third aspect of the invention, wherein the nucleicacid species, compositions, polypeptides, vaccines are provided asseparate components.

In embodiments, the combination comprises 2, 3, 4 or 5 nucleic acidspecies (e.g. DNA or RNA) comprised in separate components, preferablyRNA species, wherein said nucleic acid species comprise or consist of anucleic acid sequence which is identical or at least 70%, 80%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to a nucleic acid sequence selected from the group consistingof SEQ ID NOs: 116-132, 134-138, 140-143, 145-175, 11664-11813, 11815,11817-12050, 12052, 12054-13147, 13514, 13515, 13519, 13520,14124-14177, 22759, 22764-22786, 22791-22813, 22818-22839, 22969-23184,23189-23404, 23409-23624, 23629-23844, 23849-24064, 24069-24284,24289-24504, 24509-24724, 24729-24944, 24949-25164, 25169-25384,25389-25604, 25609-25824, 25829-26044, 26049-26264, 26269-26484,26489-26704, 26709-26937 and, optionally, at least one pharmaceuticallyacceptable carrier or excipient, wherein each of the 2, 3, 4 or 5nucleic acid species encode a different antigenic peptide or protein ofa SARS-CoV-2 coronavirus.

Accordingly, in embodiments, the combination comprises two nucleic acidspecies (e.g. DNA or RNA)) comprised in separate components, preferablyRNA species, wherein the nucleic acid species comprise or consist of anucleic acid sequence which is identical or at least 70%, 80%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to a nucleic acid sequence selected from the group consistingof SEQ ID NOs: 148-175, 12204-13147, 14142-14177, 22786-22839,23189-23404, 23409-23624, 23629-23844, 23849-24064, 24069-24284,24289-24504, 24509-24724, 24729-24944, 24949-25164, 25169-25384,25389-25604, 25609-25824, 25829-26044, 26049-26264, 26269-26484,26489-26704, 26709-26937148 and, optionally, at least onepharmaceutically acceptable carrier or excipient, wherein each of thetwo nucleic acid species encode a different antigenic peptide or proteinof a SARS-CoV-2 coronavirus.

In embodiments, the combination comprises three nucleic acid species(e.g. DNA or RNA)) comprised in separate components, preferably RNAspecies, wherein the nucleic acid comprises or consists of a nucleicacid sequence which is identical or at least 70%, 80%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identicalto a nucleic acid sequence selected from the group consisting of SEQ IDNOs: 148-175, 12204-13147, 14142-14177, 22786-22839, 23189-23404,23409-23624, 23629-23844, 23849-24064, 24069-24284, 24289-24504,24509-24724, 24729-24944, 24949-25164, 25169-25384, 25389-25604,25609-25824, 25829-26044, 26049-26264, 26269-26484, 26489-26704,26709-26937 and, optionally, at least one pharmaceutically acceptablecarrier or excipient, wherein each of the 2, 3, 4 or 5 nucleic acidspecies encode a different antigenic peptide or protein of a SARS-CoV-2coronavirus.

In the following, particularly preferred embodiments of a combinationare provided, wherein each component of the combination is provided asseparate entities.

Preferably, the at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or even moredifferent nucleic acid species, compositions, vaccines of thecombination each encode a different prefusion stabilized spike protein(as defined in the first aspect). Preferably, stabilization of theperfusion conformation is obtained by introducing two consecutiveproline substitutions at residues K986 and V987 in the spike protein(Amino acid positions according to reference SEQ ID NO: 1). Accordingly,in preferred embodiments, the at least 2, 3, 4, 5, 6, 7, 8, 9, 10pre-fusion stabilized spike proteins (S_stab) each comprises at leastone pre-fusion stabilizing mutation, wherein the at least one pre-fusionstabilizing mutation comprises the following amino acid substitutions:K986P and V987P (amino acid positions according to reference SEQ ID NO:1).

Accordingly, the at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or even moredifferent nucleic acid species, compositions, vaccines of thecombination each encode a different prefusion stabilized spike protein,wherein the at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or even more stabilizedspike proteins are selected from amino acid sequences being identical orat least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 10-26,341-407, 609-1278, 13521-13587, 22738, 22740, 22742, 22744, 22746,22748, 22750, 22752, 22754, 22756, 22758, 22947-2296410 or animmunogenic fragment or immunogenic variant of any of these.

In preferred embodiments, the combination comprises one nucleic acidspecies, composition, vaccine comprising a coding sequence encoding anamino acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identicalto any one of SEQ ID NOs: 10, wherein the multivalent compositionadditionally comprises at least 2, 3, 4 further RNA species selectedfrom

-   i) one nucleic acid species comprises a coding sequence encoding an    amino acid sequence being identical or at least 70%, 80%, 85%, 86%,    87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%    identical to any one of SEQ ID NOs: 22961; and/or-   ii) one nucleic acid species comprises a coding sequence encoding an    amino acid sequence being identical or at least 70%, 80%, 85%, 86%,    87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%    identical to any one of SEQ ID NOs: 22960; and/or-   iii) one nucleic acid species comprises a coding sequence encoding    an amino acid sequence being identical or at least 70%, 80%, 85%,    86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or    99% identical to any one of SEQ ID NOs: 22963; and/or-   iv) one nucleic acid species comprises a coding sequence encoding an    amino acid sequence being identical or at least 70%, 80%, 85%, 86%,    87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%    identical to any one of SEQ ID NOs: 22941; and/or-   v) one nucleic acid species comprises a coding sequence encoding an    amino acid sequence being identical or at least 70%, 80%, 85%, 86%,    87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%    identical to any one of SEQ ID NOs: 22964.

Preferably, the at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or even moredifferent nucleic acid species, composition, vaccine of the combinationcomprise nucleic acid coding sequences each encoding a differentprefusion stabilized spike protein, wherein the at least 2, 3, 4, 5, 6,7, 8, 9, 10 or even more nucleic acid coding sequences are selected fromnucleic acid sequences being identical or at least 70%, 80%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to any one of SEQ ID NOs: 136-138, 140-143, 145-175,11731-11813, 11815, 11817-12050, 12052, 12054-12203, 13514, 13515,13519, 13520, 14124-14141, 22759, 22764-22785, 22969-23184 or fragmentsor variants of any of these.

In preferred embodiments, the combination comprises one nucleic acidspecies, composition, vaccine comprising a coding sequence beingidentical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs:137, wherein the multivalent composition additionally comprises at least2, 3, 4 further RNA species selected from

-   i) one nucleic acid species comprises a coding sequence being    identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,    92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of    SEQ ID NOs: 23091; and/or-   ii) one nucleic acid species comprises a coding sequence being    identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,    92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of    SEQ ID NOs: 23090; and/or-   iii) one nucleic acid species comprises a coding sequence being    identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,    92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of    SEQ ID NOs: 23093; and/or-   iv) one nucleic acid species comprises a coding sequence being    identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,    92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of    SEQ ID NOs: 22999; and/or-   v) one nucleic acid species comprises a coding sequence being    identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,    92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of    SEQ ID NOs: 23094.

Preferably, the at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or even moredifferent nucleic acid species, compositions, vaccines of thecombination comprise nucleic acid coding sequences each encoding adifferent prefusion stabilized spike protein, wherein the at least 2, 3,4, 5, 6, 7, 8, 9, 10 or even more nucleic acid coding sequences areselected from RNA sequences being identical or at least 70%, 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to any one of SEQ ID NOs: 149-151, 163-165, 12338, 12541,12810-12813, 12901, 12931, 13013, 22792, 22794, 22796, 22798, 22802,22804, 22806, 22810, 22813, 22819, 22821, 22823, 22825, 22827, 22829,22831, 22833, 22835, 22837, 22839, 23297-23314, 23369, 23517-23520,23523-23525, 23527, 23529, 23530, 23589, 23737, 23957, 24397, 24837,25057, 25277, 25717, 26925-26937149 or fragments or variants of any ofthese.

In preferred embodiments, the combination comprises one RNA species,compositions, vaccines comprising or consisting of an RNA sequence beingidentical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs:163, wherein the combination additionally comprises at least 2, 3, 4further RNA species selected from

-   i) one RNA species comprising or consisting of an RNA sequence being    identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,    92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of    SEQ ID NOs: 23311; and/or-   ii) one RNA species comprises a coding sequence being identical or    at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,    95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs:    23310; and/or-   iii) one RNA species comprises a coding sequence being identical or    at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,    95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs:    23313; and/or-   iv) one RNA species comprises a coding sequence being identical or    at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,    95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs:    23219; and/or-   v) one RNA species comprises a coding sequence being identical or at    least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,    95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs:    23314;

wherein, preferably, each of the mRNA species comprise a Cap1 structure,and, optionally, each of the mRNA species do not comprise modifiednucleotides.

In preferred embodiments, the combination comprises one RNA species,composition, vaccine comprising or consisting of an RNA sequence beingidentical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs:149 or 24837, wherein the combination additionally comprises at least 2,3, 4 further RNA species, selected from

-   i) one RNA species comprising or consisting of an RNA sequence being    identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,    92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of    SEQ ID NOs: 23531 or 24851; and/or-   ii) one RNA species comprises a coding sequence being identical or    at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,    95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 23530    or 24850; and/or-   iii) one RNA species comprises a coding sequence being identical or    at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,    95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 23533    or 24853B; and/or-   iv) one RNA species comprises a coding sequence being identical or    at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,    95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 23439    or 24759; and/or-   v) one RNA species comprises a coding sequence being identical or at    least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,    95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 23534    or 24854;

wherein, preferably, each of the mRNA species comprise a Cap1 structure,and, optionally, each of the mRNA species do not comprise modifiednucleotides.

In a specific embodiment, a first component of the combination comprisesa viral vector vaccine/composition, such as an adenovirus vector basedvaccine, e.g., ADZ1222 or Ad26.COV-2.S, and a second component comprisesa nucleic acid based vaccine/composition, preferably an mRNA-basedvaccine as defined herein.

First and Second/Further Medical Use:

A further aspect relates to the first medical use of the providednucleic acid, composition, polypeptide, vaccine, kit, or combination.

Notably, embodiments relating to the nucleic acid of the first aspect,the composition of the second aspect, the polypeptide of the thirdaspect, and the vaccine of the fourth aspect, or the kit or kit of partsof the fifth aspect, or the combination may likewise be read on and beunderstood as suitable embodiments of medical uses of the invention.

Accordingly, the invention provides at least one nucleic acid (e.g. DNAor RNA), preferably RNA as defined in the first aspect for use as amedicament, the composition as defined in the second aspect for use as amedicament, the polypeptide as defined in the third aspect for use as amedicament, the vaccine as defined in the fourth aspect for use as amedicament, and the kit or kit of parts as defined in the fifth aspectfor use as a medicament, and the combination.

The present invention furthermore provides several applications and usesof the nucleic acid, composition, polypeptide, vaccine, or kit, orcombination.

In particular, nucleic acid (preferably RNA), composition, polypeptide,vaccine, or kit, or combination may be used for human medical purposesand also for veterinary medical purposes, preferably for human medicalpurposes.

In particular, nucleic acid (preferably RNA), composition, polypeptide,vaccine, or kit or kit of parts or combination is for use as amedicament for human medical purposes, wherein said nucleic acid(preferably RNA), composition, polypeptide, vaccine, or kit or kit ofparts may be suitable for young infants, newborns, immunocompromisedrecipients, as well as pregnant and breast-feeding women and elderlypeople. In particular, nucleic acid (preferably RNA), composition,polypeptide, vaccine, or kit or kit of parts is for use as a medicamentfor human medical purposes, wherein said nucleic acid (preferably RNA),composition, polypeptide, vaccine, or kit or kit of parts isparticularly suitable for elderly human subjects.

Said nucleic acid (preferably RNA), composition, polypeptide, vaccine,or kit or combination is for use as a medicament for human medicalpurposes, wherein said RNA, composition, vaccine, or the kit or kit ofparts may be particularly suitable for intramuscular injection orintradermal injection.

In yet another aspect, the invention relates to the second medical useof the provided nucleic acid, composition, polypeptide, vaccine, or kitor combination.

Accordingly, the invention provides at least one nucleic acid,preferably RNA as defined in the first aspect for treatment orprophylaxis of an infection with a coronavirus, preferably SARS-CoV-2coronavirus, or a disorder or a disease related to such an infection,such as COVID-19; a composition as defined in the second aspect fortreatment or prophylaxis of an infection with a coronavirus, preferablySARS-CoV-2 coronavirus, or a disorder or a disease related to such aninfection, such as COVID-19; a polypeptide as defined in the thirdaspect for treatment or prophylaxis of an infection with a coronavirus,preferably SARS-CoV-2 coronavirus, or a disorder or a disease related tosuch an infection, such as COVID-19; a vaccine as defined in the fourthaspect for treatment or prophylaxis of an infection with a coronavirus,preferably SARS-CoV-2 coronavirus, or a disorder or a disease related tosuch an infection, such as COVID-19; a kit or kit of parts as defined inthe fifth aspect for treatment or prophylaxis of an infection with acoronavirus, preferably SARS-CoV-2 coronavirus, or a disorder or adisease related to such an infection, such as COVID-19; a combination asdefined in the sixth aspect for treatment or prophylaxis of an infectionwith a coronavirus, preferably SARS-CoV-2 coronavirus, or a disorder ora disease related to such an infection, such as COVID-19.

In embodiments, the nucleic acid, preferably RNA of the first aspect,the composition of the second aspect, the polypeptide of the thirdaspect, the vaccine of the fourth aspect, or the kit or kit of parts ofthe fifth aspect, or the combination of the sixth aspect, is for use inthe treatment or prophylaxis of an infection with a coronavirus,preferably with SARS-CoV-2 coronavirus.

Particularly, the nucleic acid, preferably RNA of the first aspect, thecomposition of the second aspect, the polypeptide of the third aspect,the vaccine of the fourth aspect, or the kit or kit of parts of thefifth aspect, or the combination of the sixth aspect, may be used in amethod of prophylactic (pre-exposure prophylaxis or post-exposureprophylaxis) and/or therapeutic treatment of infections caused by acoronavirus, preferably SARS-CoV-2 coronavirus.

Particularly, the nucleic acid, preferably RNA of the first aspect, thecomposition of the second aspect, the polypeptide of the third aspect,the vaccine of the fourth aspect, or the kit or kit of parts of thefifth aspect, or the combination of the sixth aspect, may be used in amethod of prophylactic (pre-exposure prophylaxis or post-exposureprophylaxis) and/or therapeutic treatment of COVID-19 disease caused bya SARS-CoV-2 coronavirus infection.

The nucleic acid, the composition, the polypeptide, or the vaccine, orthe combination may preferably be administered locally. In particular,composition or polypeptides or vaccines or combinations may beadministered by an intradermal, subcutaneous, intranasal, orintramuscular route. In embodiments, the inventive nucleic acid,composition, polypeptide, vaccine may be administered by conventionalneedle injection or needle-free jet injection. Preferred in that contextis intramuscular injection.

In embodiments where plasmid DNA is used and comprised in thecomposition or vaccine or combination, thecomposition/vaccine/combination may be administered by electroporationusing an electroporation device, e.g. an electroporation device forintradermal or intramuscular delivery. Suitably, a device as describedin U.S. Pat. No. 7,245,963B2 may be used, in particular a device asdefined by claims 1 to 68 of U.S. Pat. No. 7,245,963B2.

In embodiments where adenovirus DNA is used and comprised in thecomposition or vaccine or combination, thecomposition/vaccine/combination may be administered by intranasaladministration.

In embodiments, the nucleic acid as comprised in a composition orvaccine or combination as defined herein is provided in an amount ofabout 100 ng to about 500 ug, in an amount of about 1 ug to about 200ug, in an amount of about 1 ug to about 100 ug, in an amount of about 5ug to about 100 ug, preferably in an amount of about 10 ug to about 50ug, specifically, in an amount of about 1 ug, 2 ug, 3 ug, 4 ug, 5 ug, 8ug, 9 ug, 10 ug, 11 ug, 12 ug, 13 ug, 14 ug, 15 ug, 16 ug 20 ug, 25 ug,30 ug, 35 ug, 40 ug, 45 ug, 50 ug, 55 ug, 60 ug, 65 ug, 70 ug, 75 ug, 80ug, 85 ug, 90 ug, 95 ug or 100 ug.

In some embodiments, the vaccine comprising the nucleic acid, or thecomposition comprising the nucleic acid is formulated in an effectiveamount to produce an antigen specific immune response in a subject. Insome embodiments, the effective amount of nucleic acid is a total doseof 1 ug to 200 ug, 1 ug to 100 ug, or 5 ug to 100 ug.

In embodiments where the nucleic acid is provided in a lipid-basedcarrier, e.g. an LNP, the amount of PEG-lipid as defined hereincomprised in one dose is lower than about 50 μg PEG lipid, preferablylower than about 45 μg PEG lipid, more preferably lower than about 40 μgPEG lipid.

Having a low amount of PEG lipid in one dose may reduce the risk ofadverse effects (e.g. allergies).

In particularly preferred embodiments, the amount of PEG-lipid comprisedin one dose is in a range from about 3.5 μg PEG lipid to about 35 μg PEGlipid.

In embodiments where the nucleic acid is provided in a lipid-basedcarrier, e.g. an LNP, the amount of cationic lipid as defined hereincomprised in one dose is lower than about 400 μg cationic lipid,preferably lower than about 350 μg cationic lipid, more preferably lowerthan about 300 μg cationic lipid.

Having a low amount of cationic lipid in one dose may reduce the risk ofadverse effects (e.g. fewer).

In particularly preferred embodiments, the amount of cationic-lipidcomprised in one dose is in a range from about 30 μg PEG lipid to about300 μg PEG lipid.

In one embodiment, the immunization protocol for the treatment orprophylaxis of a subject against coronavirus, preferably SARS-CoV-2coronavirus comprises one single doses of the composition or thevaccine.

In some embodiments, the effective amount is a dose of 1 ug administeredto the subject in one vaccination. In some embodiments, the effectiveamount is a dose of 2 ug administered to the subject in one vaccination.In some embodiments, the effective amount is a dose of 3 ug administeredto the subject in one vaccination. In some embodiments, the effectiveamount is a dose of 4 ug administered to the subject in one vaccination.In some embodiments, the effective amount is a dose of 5 ug administeredto the subject in one vaccination. 6 ug administered to the subject inone vaccination. In some embodiments, the effective amount is a dose of7 ug administered to the subject in one vaccination. In someembodiments, the effective amount is a dose of 8 ug administered to thesubject in one vaccination. In some embodiments, the effective amount isa dose of 9 ug administered to the subject in one vaccination. In someembodiments, the effective amount is a dose of 10 ug administered to thesubject in one vaccination. In some embodiments, the effective amount isa dose of 11 ug administered to the subject in one vaccination. In someembodiments, the effective amount is a dose of 12 ug administered to thesubject in one vaccination. In some embodiments, the effective amount isa dose of 13 ug administered to the subject in one vaccination. In someembodiments, the effective amount is a dose of 14 ug administered to thesubject in one vaccination. In some embodiments, the effective amount isa dose of 16 ug administered to the subject in one vaccination. In someembodiments, the effective amount is a dose of 20 ug administered to thesubject in one vaccination. In some embodiments, the effective amount isa dose of 25 ug administered to the subject in one vaccination. In someembodiments, the effective amount is a dose of 30 ug administered to thesubject in one vaccination. In some embodiments, the effective amount isa dose of 40 ug administered to the subject in one vaccination. In someembodiments, the effective amount is a dose of 50 ug administered to thesubject in one vaccination. In some embodiments, the effective amount isa dose of 100 ug administered to the subject in one vaccination. In someembodiments, the effective amount is a dose of 200 ug administered tothe subject in one vaccination. A “dose” in that context relates to theeffective amount of nucleic acid, preferably mRNA as defined herein.

In preferred embodiments, the immunization protocol for the treatment orprophylaxis of a coronavirus, preferably a SARS-CoV-2 coronavirusinfection comprises a series of single doses or dosages of thecomposition or the vaccine. A single dosage, as used herein, refers tothe initial/first dose, a second dose or any further doses,respectively, which are preferably administered in order to “boost” theimmune reaction.

In some embodiments, the effective amount is a dose of 1 ug administeredto the subject a total of two times. In some embodiments, the effectiveamount is a dose of 2 ug administered to the subject a total of twotimes. In some embodiments, the effective amount is a dose of 3 ugadministered to the subject a total of two times. In some embodiments,the effective amount is a dose of 4 ug administered to the subject atotal of two times. In some embodiments, the effective amount is a doseof 5 ug administered to the subject a total of two times. In someembodiments, the effective amount is a dose of 6 ug administered to thesubject a total of two times. In some embodiments, the effective amountis a dose of 7 ug administered to the subject a total of two times. Insome embodiments, the effective amount is a dose of 8 ug administered tothe subject a total of two times. In some embodiments, the effectiveamount is a dose of 9 ug administered to the subject a total of twotimes. In some embodiments, the effective amount is a dose of 10 ugadministered to the subject a total of two times. In some embodiments,the effective amount is a dose of 11 ug administered to the subject atotal of two times. In some embodiments, the effective amount is a doseof 12 ug administered to the subject a total of two times. In someembodiments, the effective amount is a dose of 13 ug administered to thesubject a total of two times. In some embodiments, the effective amountis a dose of 14 ug administered to the subject a total of two times. Insome embodiments, the effective amount is a dose of 16 ug administeredto the subject a total of two times. In some embodiments, the effectiveamount is a dose of 20 ug administered to the subject a total of twotimes. In some embodiments, the effective amount is a dose of 25 ugadministered to the subject a total of two times. In some embodiments,the effective amount is a dose of 30 ug administered to the subject atotal of two times. In some embodiments, the effective amount is a doseof 40 ug administered to the subject a total of two times. In someembodiments, the effective amount is a dose of 50 ug administered to thesubject a total of two times. In some embodiments, the effective amountis a dose of 100 ug administered to the subject a total of two times. Insome embodiments, the effective amount is a dose of 200 ug administeredto the subject a total of two times. A “dose” in that context relates tothe effective amount of nucleic acid, preferably mRNA as defined herein.

In preferred embodiments, the vaccine/composition/combination immunizesthe subject against a coronavirus, preferably against a SARS-CoV-2coronavirus infection (upon administration as defined herein) for atleast 1 year, preferably at least 2 years. In preferred embodiments, thevaccine/composition/combination immunizes the subject against acoronavirus, preferably against a SARS-CoV-2 coronavirus for more than 2years, more preferably for more than 3 years, even more preferably formore than 4 years, even more preferably for more than 5-10 years.

Method of Treatment and Use, Diagnostic Method and Use:

In another aspect, the present invention relates to a method of treatingor preventing a disorder.

Notably, embodiments relating to the nucleic acid of the first aspect,the composition of the second aspect, the polypeptide of the thirdaspect, and the vaccine of the fourth aspect, the kit or kit of parts ofthe fifth aspect, the combination of the sixth aspect, or medical usesmay likewise be read on and be understood as suitable embodiments ofmethods of treatments as provided herein. Furthermore, specific featuresand embodiments relating to method of treatments as provided herein mayalso apply for medical uses of the invention.

Preventing (Inhibiting) or treating a disease, in particular acoronavirus infection relates to inhibiting the full development of adisease or condition, for example, in a subject who is at risk for adisease such as a coronavirus infection. “Treatment” refers to atherapeutic intervention that ameliorates a sign or symptom of a diseaseor pathological condition after it has begun to develop. The term“ameliorating”, with reference to a disease or pathological condition,refers to any observable beneficial effect of the treatment. Inhibitinga disease can include preventing or reducing the risk of the disease,such as preventing or reducing the risk of viral infection. Thebeneficial effect can be evidenced, for example, by a delayed onset ofclinical symptoms of the disease in a susceptible subject, a reductionin severity of some or all clinical symptoms of the disease, a slowerprogression of the disease, a reduction in the viral load, animprovement in the overall health or well-being of the subject, or byother parameters that are specific to the particular disease. A“prophylactic” treatment is a treatment administered to a subject whodoes not exhibit signs of a disease or exhibits only early signs for thepurpose of decreasing the risk of developing pathology.

In preferred embodiments, the present invention relates to a method oftreating or preventing a disorder, wherein the method comprises applyingor administering to a subject in need thereof at least one nucleic acidof the first aspect, the composition of the second aspect, thepolypeptide of the third aspect, the vaccine of the fourth aspect, orthe kit or kit of parts of the fifth aspect, or the combination of thesixth aspect.

In preferred embodiments, the disorder is an infection with acoronavirus, or a disorder related to such infections, in particular aninfection with SARS-CoV-2 coronavirus, or a disorder related to suchinfections, e.g. COVID-19.

In preferred embodiments, the present invention relates to a method oftreating or preventing a disorder as defined above, wherein the methodcomprises applying or administering to a subject in need thereof atleast one nucleic acid of the first aspect, the composition of thesecond aspect, the polypeptide of the third aspect, the vaccine of thefourth aspect, or the kit or kit of parts of the fifth aspect, or thecombination of the sixth aspect, wherein the subject in need ispreferably a mammalian subject.

In certain embodiments, a method of treating or preventing disease byapplying or administering to a subject in need thereof at least onenucleic acid of the first aspect, the composition of the second aspect,the polypeptide of the third aspect, the vaccine of the fourth aspect,or the kit or kit of parts of the fifth aspect or the combination of thesixth aspect, is further defined as a method of reducing disease burdenin the subject. For example, the method preferably reduces the severityand/or duration of one or more symptom of COVID-19 disease. In someaspects, a method reduces the probability that a subject will requirehospital admission, intensive care unit admission, treatment withsupplemental oxygen and/or treatment with a ventilator. In furtheraspects, the method reduces the probability that a subject will developa fever, breathing difficulties; loss of smell and/or loss of taste. Inpreferred aspects, the method reduces the probability that a subjectwill develop severe or moderate COVID-19 disease. In certain aspects, amethod of the embodiments prevents severe or moderate COVID-19 diseasein the subject between about 2 weeks and 1 month, 2 months, 3 months, 4months, 5 months, 6 months, 1 year or 2 years after the subject isadministered a composition of the embodiments. In preferred aspects, amethod of the embodiments prevents symptomatic COVID-19 disease. Infurther aspects, a method of the embodiment prevents detectable levelsof SARS CoV-2 nucleic acid in the subject between about 2 weeks and 1month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year or 2years after the subject is administered a composition of theembodiments. In further aspects, a method of the embodiments is definedas a method for providing protective immunity to a coronavirus infection(e.g., SARS CoV-2 infection) in the subject. In still further aspects, amethod of the embodiments prevents moderate and severe COVID-19 diseasein at least 80%, 85%, 90% or 95% of treated subjects. In yet furtheraspects, a method of the embodiments prevents moderate and severeCOVID-19 disease in at least 80%, 85%, 90% or 95% of treated subjectsfrom about 2 weeks to about 1 year after administering the second orsubsequent immunogenic composition (e.g., a booster administration). Inyet further aspects, a method of the embodiments prevents moderate andsevere COVID-19 disease in at least 80%, 85%, 90% or 95% of treatedsubjects from about 2 weeks to about 3 month, 6 months, 9 months, 1year, 1.5 years, 2 years or 3 years after administering the second orsubsequent composition.

In a further aspects, a method of the embodiments comprises (i)obtaining a composition (e.g., a vaccine composition) of theembodiments, wherein the composition is lyophilized; (ii) solubilizingthe lyophilized composition in a pharmaceutically acceptable liquidcarrier to produce a liquid composition; and (iii) administering aneffective amount of the liquid composition to the subject. In someaspects, the lyophilized composition comprises less than about 10% watercontent. For example, the lyophilized composition can preferablycomprise about 0.1% to about 10%, 0.5% to 7.5% or 0.5% to 5.0% water.

In still further aspects, a method of the embodiments comprisesadministering a vaccine composition comprising a mRNA at least about 95%identical to SEQ ID NO: 163 (e.g., in complex with a LNP) to a subject.

In further aspects, a method of the embodiments comprises administeringa vaccine composition comprising a mRNA at least about 95% identical toSEQ ID NO: 149, 24837, 23311, 23531, 23310, 23530, 23313, or 23533(e.g., in complex with a LNP) to a subject. In some aspects, such amethod provides a sufficient immune response in the subject to protectthe subject from severe COVID-19 disease for at least about 6 months.For example, in some aspects, the subject is protected from severeCOVID-19 disease for about 6 months to about 1 year, 1.5 years, 2 years,2.5 years, 3 years, 4 years or 5 years. Thus, in some aspects, a methodof the embodiments provides a single dose vaccine composition that canprovide prolonged (e.g., greater than 6 months of) protection fromsevere disease to a subject.

As used herein severe COVID-19 disease is defined as a subjectexperiencing one or more of the following:

-   -   Clinical signs at rest indicative of severe systemic illness        (respiratory rate≥30 breaths per minute, heart rate 125 per        minute, SpO2≤93% on room air at sea level or PaO2/FIO2<300 mm Hg        (adjusted according to altitude))    -   Respiratory failure (defined as needing high flow-oxygen,        noninvasive ventilation, mechanical ventilation or ECMO)    -   Evidence of shock (SBP<90 mm Hg, DBP<60 mmHg, or requiring        vasopressors)    -   Significant renal, hepatic, or neurologic dysfunction    -   Admission to ICU    -   Death

As used herein moderate COVID-19 disease is defined as a subjectexperiencing one or more of the following:

-   -   Shortness of breath or difficulty breathing    -   Respiratory rate≥20 breaths per minute    -   Abnormal SpO2 but still >93% on room air at sea level (adjusted        according to altitude)    -   Clinical or radiographic evidence of lower respiratory tract        disease    -   Radiologic evidence of deep vein thrombosis (DVT)

As used herein mild COVID-19 disease is defined as a subjectexperiencing all of the following:

-   -   Symptomatic AND    -   No shortness of breath or difficulty breathing AND    -   No hypoxemia (adjusted according to altitude) AND    -   Does not meet the case definition of moderate or severe COVID-19        disease

In particularly preferred embodiments, the subject in need is amammalian subject, preferably a human subject, e.g. newborn, pregnant,immunocompromised, and/or elderly. In some embodiments, the subjectbetween the ages of 6 months and 100 years, 6 months and 80 years, 1year and 80 years, 1 year and 70 years, 2 years and 80 years or 2 yearsand 60 years. In other embodiments the subject is a newborn or infant ofan age of not more than 3 years, of not more than 2 years, of not morethan 1.5 years, of not more than 1 year (12 months), of not more than 9months, 6 months or 3 months. In some other embodiments the subject isan elderly subject of an age of at least 50, 60, 65, or 70 years. Infurther aspects, a subject for treatment according to the embodiments is61 years of age or older. In still further aspects, the subject is 18years old to 60 years old.

In certain embodiments, a subject for treatment according to theembodiments is a pregnant subject, such a pregnant human. In someaspects, the subject has been pregnant for more than about one month,two months, three months, four months, five months, six months, sevenmonths or eight months.

In particularly preferred embodiments, the human subject is an elderlyhuman subject.

In certain aspects, a subject for treatment according to the embodimentshas native American, African, Asian or European heritage. In someaspects, the subject has at least about 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% native American,African, Asian or European heritage. In certain aspects, the subject hasnative American heritage, such as at least about 10%, 25% or 50% nativeAmerican heritage. In further aspects, the subject is an elderly subjecthaving native American heritage, e.g., a subject who is at least 55, 60,65 or 70 years of age.

In further aspects, a subject for treatment according to the embodimentshas a disease or is immune compromised. In some aspects, the subject hasliver disease, kidney disease diabetes, hypertension, heart disease orlung disease. In further aspects, a subject for treatment according tothe embodiments is a subject with history of allergic reaction, such asubject having food allergies. In some aspect, the subject has had aprevious allergic reaction to a vaccine, such as an anaphylacticreaction. In still further aspects, a subject for treatment according tothe methods is a subject having detectable anti-PEG antibodies, such asdetectable anti-PEG IgE in the serum.

In further aspects, a subject for treatment according to the embodimentshas at least one co-morbidity selected from:

(i) Chronic kidney disease: Kidney function will be ascertained from theserum creatinine measurement within the last 3-6 months, converted intoestimated glomerular filtration rate (eGFR) using the Chronic KidneyDisease Epidemiology Collaboration (CKD-EPI) equation, with impairedkidney function defined as eGFR<60 mL/min/1.73 m².

-   -   Mild chronic kidney disease is defined as an eGFR between 60 89        mL/min/1.73 m2.    -   Moderate chronic kidney disease is defined as an eGFR between        31-59 mL/min/1.73 m2 with stable therapy and good maintenance        over at least 6 months (modified from Clinical Practice Clinical        Guidelines for Chronic Kidney Disease: Am J Kidney Dis, 2002).

(ii) COPD (including emphysema and chronic bronchitis).

-   -   Mild COPD with or without cough or sputum production is defined        as forced expiratory volume in 1 second/forced vital capacity        (FEV1/FVC)≤0.7 and FEV1≥80% predicted.    -   Moderate COPD with or without cough or sputum production is        defined as FEV1/FVC<0.7 and FEV1≥50%, but <80% predicted with        stable treatment (GOLD Criteria for COPD severity).

(iii) Obesity with body mass index (BMI) of >32 kg/m²—any extreme morbidobesity will also be included.

(iv) Chronic cardiovascular conditions (heart failure, coronary arterydisease, cardiomyopathies, arterial hypertension), including thefollowing:

-   -   Class I heart failure with potential high risk for developing        heart failure in future with no functional or structural heart        disorder.    -   Class II heart failure: subjects with cardiac disease resulting        in slight limitation of physical activity. Comfortable at rest.    -   Ordinary physical activity results in fatigue, palpitation,        dyspnea, or anginal pain.    -   Class III heart failure with marked limitation of physical        activity, but comfortable at rest but less than ordinary        activity results in symptoms.    -   A structural heart disorder without symptoms at any stage.    -   Mild left ventricular systolic or diastolic dysfunction, usually        with not much produced clinical signs.    -   Moderate left ventricular failure with exertional dyspnea or        orthopnea or paroxysmal nocturnal dyspnea as per New York Heart        Association (NYHA), stable with medication (Class    -   Coronary artery disease of 2 and above metabolic equivalent        threshold (MET) up to moderate, stable with medication. (MET is        defined as the amount of oxygen consumed while sitting at rest        and is equal to 3.5 ml O₂ per kg body weight×min; 4 Normal, can        climb a flight of stairs or walk up a hill and can participate        in other strenuous activities; 1 can take care of him/herself        and may not maintain themselves and gets constraints on        exertion.)    -   Cardiomyopathies of non-infective and metabolic origin of 2-3        MET with medication.    -   Stage 1 hypertension or Stage 2 hypertension stable and        controlled with medications.

(v) Chronic HIV infection with stable aviraemia (<50 copies/mL) and CD4count >350/mL as documented by blood samples taken within 12 monthsbefore enrolment. (Viral load<50 copies/mL with transient changes of50-350 copies/mL is allowed.)

(vi) Type 2 diabetes mellitus, either controlled with medication[hemoglobin A1c (HbA1c)<58 mmol/mol (7.45%)] or uncontrolled with recentHbA1c of >58 mmol/mol (7.45%); [(HbA1c in %−2.15)×10.929=HbA1c inmmol/mol]; in uncontrolled DM HbA1c should be within <10% variation andshould not have any history of diabetic ketoacidosis or episode ofsevere symptomatic hypoglycemia within prior 3 months.

(vii) Subjects who underwent renal transplant at least a year ago understable conditions for at least 6 months with medications, categorized aslow risk of rejection.

In still further aspects, a subject for treatment according to theembodiments has not been treated with an immunosuppressant drug for morethan 14 days in the last 6 months. In some aspects, a subject fortreatment according to the embodiments has not received a live vaccinefor at least 28 days prior to the administration and/or has not receivedan inactivated vaccine for at least 14 days prior to the administration.In further aspects a subject for treatment according to the embodimentshas NOT:

-   -   Had virologically-confirmed COVID-19 illness;    -   For females: experienced pregnancy or lactation with-in a month        prior to administration of the composition of the embodiments;    -   had treatment with an investigational or non-registered product        (e.g., vaccine or drug) within 28 days preceding the        administration of the composition of the embodiments;    -   received a licensed vaccines within 28 days (for live vaccines)        or 14 days (for inactivated vaccines) prior to the        administration of the composition of the embodiments;    -   been previously or concurrently treated with any investigational        SARS-CoV-2 vaccine or another coronavirus (SARS-CoV, MERS-CoV)        vaccine;    -   been treated with immunosuppressants or other immune-modifying        drugs (e.g., corticosteroids, biologicals and methotrexate)        for >14 days total within 6 months preceding the administration        of the composition of the embodiments;    -   had any medically diagnosed or suspected immunosuppressive or        immunodeficient condition based on medical history and physical        examination including known infection with human        immunodeficiency virus (HIV), hepatitis B virus (HBV) or        hepatitis C virus (HCV); current diagnosis of or treatment for        cancer including leukemia, lymphoma, Hodgkin disease, multiple        myeloma, or generalized malignancy; chronic renal failure or        nephrotic syndrome; and receipt of an organ or bone marrow        transplant.    -   had a history of angioedema (hereditary or idiopathic), or        history of any anaphylactic reaction or pIMD.    -   a history of allergy to any component of CVnCoV vaccine.    -   been administered of immunoglobulins or any blood products        within 3 months prior to the administration of the composition        of the embodiments;    -   experienced a significant acute or chronic medical or        psychiatric illness; and/or    -   experienced severe and/or uncontrolled cardiovascular disease,        gastrointestinal disease, liver disease, renal disease,        respiratory disease, endocrine disorder, and neurological and        psychiatric illnesses.

In certain aspects, a subject for treatment according to the methods ofthe embodiments does not have any potential immune-mediated disease(pIMD). In further aspects, a treatment method of the embodiments doesnot induce any pIMD in a treated subject. As used herein pIMDs aredefined as Celiac disease; Crohn's disease; Ulcerative colitis;Ulcerative proctitis; Autoimmune cholangitis; Autoimmune hepatitis;Primary biliary cirrhosis; Primary sclerosing cholangitis; Addison'sdisease; Autoimmune thyroiditis (including Hashimoto thyroiditis;Diabetes mellitus type I; Grave's or Basedow's disease; Antisynthetasesyndrome; Dermatomyositis; Juvenile chronic arthritis (including Still'sdisease); Mixed connective tissue disorder; Polymyalgia rheumatic;Polymyositis; Psoriatic arthropathy; Relapsing polychondritis;Rheumatoid arthritis; Scleroderma, (e.g., including diffuse systemicform and CREST syndrome); Spondyloarthritis, (e.g., including ankylosingspondylitis, reactive arthritis (Reiter's Syndrome) and undifferentiatedspondyloarthritis); Systemic lupus erythematosus; Systemic sclerosis;Acute disseminated encephalomyelitis, (including site specific variants(e.g., non-infectious encephalitis, encephalomyelitis, myelitis,myeloradiculomyelitis)); Cranial nerve disorders, (e.g., includingparalyses/paresis (e.g., Bell's palsy)); Guillain-Barré syndrome, (e.g.,including Miller Fisher syndrome and other variants); Immune-mediatedperipheral neuropathies, Parsonage-Turner syndrome and plexopathies,(e.g., including chronic inflammatory demyelinating polyneuropathy,multifocal motor neuropathy, and polyneuropathies associated withmonoclonal gammopathy); Multiple sclerosis; Narcolepsy; Optic neuritis;Transverse Myelitis; Alopecia areata; Autoimmune bullous skin diseases,including pemphigus, pemphigoid and dermatitis herpetiformis; Cutaneouslupus erythematosus; Erythema nodosum; Morphoea; Lichen planus;Psoriasis; Sweet's syndrome; Vitiligo; Large vessels vasculitis (e.g.,including: giant cell arteritis such as Takayasu's arteritis andtemporal arteritis); Medium sized and/or small vessels vasculitis (e.g.,including: polyarteritis nodosa, Kawasaki's disease, microscopicpolyangiitis, Wegener's granulomatosis, Churg-Strauss syndrome (allergicgranulomatous angiitis), Buerger's disease thromboangiitis obliterans,necrotizing vasculitis and anti-neutrophil cytoplasmic antibody (ANCA)positive vasculitis (type unspecified), Henoch-Schonlein purpura,Behcet's syndrome, leukocytoclastic vasculitis); Antiphospholipidsyndrome; Autoimmune hemolytic anemia; Autoimmune glomerulonephritis(including IgA nephropathy, glomerulonephritis rapidly progressive,membranous glomerulonephritis, membranoproliferative glomerulonephritis,and mesangioproliferative glomerulonephritis); Autoimmunemyocarditis/cardiomyopathy; Autoimmune thrombocytopenia; Goodpasturesyndrome; Idiopathic pulmonary fibrosis; Pernicious anemia; Raynaud'sphenomenon; Sarcoidosis; Sjögren's syndrome; Stevens-Johnson syndrome;Uveitis).

In certain aspects, a vaccination method of the embodiments does notresult in a subject experiencing any adverse events of special interest(AESIs). As used herein AESIs are defined as a pIMD listed above;Anaphylaxis; Vasculitides; Enhanced disease following immunization;Multisystem inflammatory syndrome in children; Acute RespiratoryDistress Syndrome; COVID-19 disease; Acute cardiac injury;Microangiopathy; Heart failure and cardiogenic shock; Stresscardiomyopathy; Coronary artery disease; Arrhythmia; Myocarditis,pericarditis; Thrombocytopenia; Deep vein thrombosis; Pulmonary embolus;Cerebrovascular stroke; Limb ischemia; Hemorrhagic disease; Acute kidneyinjury; Liver injury; Generalized convulsion; Guillain-Barré Syndrome;Acute disseminated encephalomyelitis; Anosmia, ageusia;Meningoencephalitis; Chilblain-like lesions; Single organ cutaneousvasculitis; Erythema multiforme; Serious local/systemic AR followingimmunization

In particular, such the method of treatment may comprise the steps of:

-   a) providing at least one nucleic acid (e.g. DNA or RNA), preferably    at least one RNA of the first aspect, at least one composition of    the second aspect, at least one polypeptide of the third aspect, at    least one vaccine of the fourth aspect, or the kit or kit of parts    of the fifth aspect;-   b) applying or administering said nucleic acid, composition,    polypeptide, vaccine, or kit or kit of parts to a subject as a first    dose-   c) optionally, applying or administering said nucleic acid,    composition, polypeptide, vaccine, or kit or kit of parts to a    subject as a second dose or a further dose, preferably at least 3,    4, 5, 6, 7, 8, 9, 10, 11, 12, months after the first dose.

The first dosage, as used herein, refers to the initial/first dose, asecond dose or any further doses, respectively, which are preferablyadministered in order to “boost” the immune reaction. In certainaspects, the vaccine/composition is administered to a subject one, twothree, four or more times. In some aspects, the vaccine/composition isadministered to the subject at least first and a second time (e.g., aprime and boost). In some aspects, the send administration is at least10 days, 14 days, 21 days, 28 days, 35 days, 42 days, 49 days or 56 daysafter the first administration. In some aspects, the time between thefirst administration and the second administration is between about 7days and about 56 days; about 14 days and about 56 days; about 21 daysand about 56 days; or about 28 days and about 56 days. In furtheraspects, the vaccine/composition is administered to a subject three ormore times. In certain aspects, there is at least 10 days, 14 days, 21days, 28 days, 35 days, 42 days, 49 days or 56 days between eachadministration of the vaccine/composition.

In some aspects, a subject for treatment according to the embodimentswas previously infected with SARS CoV-2 or was previously treated withat least a first SARS CoV-2 vaccine composition. In some aspects, thesubject was treated with one, two, three or more doses of a first SARSCoV-2 vaccine composition. In some aspects, the composition of theembodiments used to treat a subject is a different type of vaccinecomposition than the composition previously used to treat the subject.In some aspects, the subject was previously treated with a mRNA vaccine,such as BNT162 or mRNA-1273. In further aspects, the subject waspreviously treated with a protein subunit vaccine, such as spike proteinbased vaccine, e.g., NVX-CoV2373 or COVAX. In certain preferred aspects,protein subunit vaccine compositions comprise an adjuvant. In furtheraspects, the subject was previously treated with a viral vector vaccine,such as an adenovirus vector based vaccine, e.g., ADZ1222 orAd26.COV-2.S. In still further aspects, the subject was previouslytreated with an inactivated virus vaccine to SARS CoV-2 such asCoronaVac, BBIBP-CorV or BBV152. In further aspects, a subjectpreviously treated with a vaccine composition has detectable SARS CoV-2binding antibodies, such as SARS CoV-2 S protein-binding antibodies orSARS CoV-2 N protein-binding antibodies. In further aspects, a subjectfor treatment according the embodiments was treated with a first SARSCoV-2 vaccine composition at least about 3 month, 6 months, 9 months, 1year, 1.5 years, 2 years or 3 years ago. In still further aspects, asubject for treatment according the embodiments was treated with a firstSARS CoV-2 vaccine composition between about 3 months and 2 years ago orbetween about 6 months and 2 years ago. In some aspects, a subjectstreated with a further vaccine composition of the embodiments areprotected from moderate and severe COVID-19 disease in at least 80%,85%, 90% or 95% of treated subjects. For example, the treated subjectscan be protected from moderate and severe COVID-19 disease in at least80%, 85%, 90% or 95% of treated subjects from about 2 weeks to about 1year after administration of the further composition. In still furtheraspects, administering the further vaccine composition of theembodiments prevents moderate and severe COVID-19 disease in at least80%, 85%, 90% or 95% of treated subjects from about 2 weeks to about 3month, 6 months, 9 months, 1 year, 1.5 years, 2 years or 3 years aftersaid administration. Examples of such combination vaccination strategiesare shown below:

Dose 1 mRNA vaccine—T1—dose 2 mRNA vaccine—T2—dose 3 mRNA vaccine

Dose 1 mRNA vaccine—T1—dose 2 mRNA vaccine—T2—dose 3 protein subunitvaccine

Dose 1 mRNA vaccine—T1—dose 2 mRNA vaccine—T2—dose 3 viral vectorvaccine

Dose 1 mRNA vaccine—T1—dose 2 mRNA vaccine—T2—dose 3 inactivated virusvaccine

Dose 1 protein subunit vaccine—T1—dose 2 protein subunit vaccine—T2—dose3 mRNA vaccine

Dose 1 inactivated virus vaccine—T1—dose 2 inactivated virusvaccine—T2—dose 3 mRNA vaccine

Dose 1 viral vector vaccine—T1—dose 2 viral vector vaccine—T2—dose 3mRNA vaccine

Dose 1 viral vector vaccine—T2—dose 2 mRNA vaccine

Dose 1 protein subunit vaccine—T2—dose 2 mRNA vaccine

Dose 1 inactivated virus vaccine—T2—dose 2 mRNA vaccine

Dose 1 mRNA vaccine—T2—dose 2 mRNA vaccine

In the examples, above time period 1 (T1) is typically 2 to 6 weeks,preferably 3 to 4 weeks. Time period 2 (T2) is in some cases, about 3months, 6 months, 9 months, 1 year, 1.5 years, 2 years or three years.

In some aspects, a method of the embodiments comprises administeringmultiple doses of a vaccine composition to a subject. In a furtheraspect, there is provided a method of reducing reactogenicity of a SARSCoV-2 booster vaccine composition. In some aspects, after an initialvaccination, subject exhibiting a high level of reactogenicity areadministered a booster vaccine that is different from the initialvaccine composition. For example, in some aspects, the initial vaccineis BNT162 or mRNA-1273 and the booster vaccine is a mRNA vaccinecomposition of the embodiments. In some aspects, a booster vaccinecomposition for a subject with high reactogenicity is selected basedhaving a lower concentration of PEG or PEG-conjugate compared to thepreviously administered vaccine composition. In some aspects, a boostervaccine composition for a subject with high reactogenicity is selectedbased on a lower concentration of mRNA or LNP compared to the previouslyadministered vaccine composition.

In certain aspects, a subject for treatment according to the embodimentsis administered a vaccine composition as booster vaccine and haspreviously been treated with one or more administrations of acoronavirus vaccine composition. In certain aspects, the subject beingtreated with a booster vaccine previously was treated with a vaccinecomposition that included a spike protein antigen or a nucleic acidmolecule encoding a spike protein antigen. In some aspects, the subjectselected for treatment with the booster vaccine was previouslyadministered a vaccine composition comprising, or encoding, a spikeprotein having a different amino acid sequence than the spike protein ofthe booster vaccine. In certain aspects, the previously administeredvaccine composition comprised, or encoded, a spike (e.g., a SARS CoV-2spike) protein having at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 aminoacid differences relative to the booster vaccine composition. In certainaspects, the booster vaccine composition comprises a RNA encoding aspike protein having about 1 to 50; about 3 to 30; about 5 to 30 orabout 10 to 25 amino acid differences relative to the previouslyadministered vaccine composition. In still further aspects, the boostervaccine composition comprises RNA encoding 2, 3, 4 or more distinctspike proteins with different amino acid sequences.

In further aspects, methods of the embodiments comprise administering 2or more booster vaccine compositions to a subject, wherein each boostervaccine composition comprises RNA encoding a distinct spike protein withdifferent amino acid sequences. In some aspects, such distinct boostervaccine compositions are administered essentially simultaneously or lessthan about 10 minutes, 20 minutes, 30 minutes, 1 hour or 2 hours apart.In some aspects, distinct booster vaccine compositions are administeredto the same site, such as intramuscular injections to the same arm ofthe subject. In further aspects, distinct booster vaccine compositionsare administered to different sites, such as intramuscular injections todifferent arms or to one or both arms and one more leg muscles.

In certain aspects, a method of the embodiments is further defined as amethod of stimulating an antibody or CD8+ T-cell response in a subject.In some aspects, the method is defined as a method of stimulating aneutralizing antibody response in a subject. In further aspects, themethod is defined as a method of stimulating a protective immuneresponse in a subject. In yet further aspects, the method is defined asa method of stimulating TH2 directed immune response in a subject.

In further aspects, administration of a vaccine/composition/combinationof the embodiments stimulates an antibody response that produces betweenabout 10 and about 500 coronavirus spike protein-binding antibodies forevery coronavirus neutralizing antibody in the subject. For example, theadministration can stimulate an antibody response that produces no morethan about 200 spike protein-binding antibodies for every coronavirusneutralizing antibody. In further aspects, the administration stimulatesan antibody response that produces between about 10 and about 300; about20 and about 300; about 20 and about 200; about 30 and about 100; orabout 30 and about 80 coronavirus spike protein-binding antibodies forevery coronavirus neutralizing antibody. In still further aspects,administration of composition of the embodiments stimulates an antibodyresponse in a subject that includes a ratio of spike protein-bindingantibodies to coronavirus neutralizing antibodies that is with 20%, 15%,10% or 5% of the ratio of spike protein-binding antibodies tocoronavirus neutralizing antibodies found in average convalescentpatient serum (from a subject who has recovered from coronavirusinfection).

In yet further aspects, administration of avaccine/composition/combination of the embodiments stimulates anantibody response that produces between about 1 and about 500coronavirus spike protein receptor binding domain (RBD)-bindingantibodies for every coronavirus neutralizing antibody in the subject.In further aspects, the administration stimulates an antibody responsethat produces no more than about 50 spike protein RBD-binding antibodiesfor every coronavirus neutralizing antibody. In still further aspects,administration stimulates an antibody response that produces betweenabout 1 and about 200; about 2 and about 100; about 3 and about 200;about 5 and about 100; about 5 and about 50; or about 5 and about 20spike protein RBD-binding antibodies for every coronavirus neutralizingantibody. In still further aspects, administration of composition of theembodiments stimulates an antibody response in a subject that includes aratio of spike protein RBD-binding antibodies to coronavirusneutralizing antibodies that is with 20%, 15%, 10% or 5% of the ratio ofspike protein RBD-binding antibodies to coronavirus neutralizingantibodies found in average convalescent patient serum (from a subjectwho has recovered from coronavirus infection).

In still further aspects, administration of avaccine/composition/combination of the embodiments induces essentiallyno increase in IL-4, IL-13, TNF and/or IL-1β in the subject. In furtheraspects, the administration of a vaccine/composition of the embodimentsinduces essentially no increase in serum IL-4, IL-13, TNF and/or IL-1βin the subject. In some aspects, the administration of avaccine/composition of the embodiments induces essentially no increasein IL-4, IL-13, TNF and/or IL-1β at the injection site (e.g., anintramuscular injection site) in the subject.

In still further aspects, a method of the embodiments comprisesadministration of a vaccine/composition of the embodiments to a humansubject having a disease. In certain aspects, the subject hascardiovascular disease, kidney disease, lung disease or an autoimmunedisease. In some aspects, a vaccine/composition of the embodiments isadministered to a subject who is receiving anti-coagulation therapy.

In still further aspects, administering avaccine/composition/combination of the embodiments to human subjectsresults in no more than 20%, 15%, 10% 7.5% or 5% of the subjectsexperiencing a Grade 3 local adverse event (see Table A below). Forexample, in some aspects, no more than 10% of subjects experience aGrade 3 local adverse event after a first or a second dose of thecomposition. In preferred aspects, administering a composition of theembodiments to human subjects results in no more than 40%, 30%, 25%,20%, 15%, 10%, 7.5% or 5% of the subjects experiencing a Grade 2 ofhigher local adverse event. For example, in some aspects, no more than30% of subjects experience a Grade 2 or higher local adverse event aftera first or a second dose of the composition. In some aspects,administering a composition of the embodiments to human subjects resultsin no more than 10% of the subjects experiencing Grade 3 pain, redness,swelling and/or itching at the injection site

In further aspects, administering a vaccine/composition/combination ofthe embodiments to human subjects results in no more than 30%, 25%, 20%,15%, 10% or 5% of the subjects experiencing a Grade 3 systemic adverseevent (see Table B below). For example, in some aspects, no more than25% of subjects experience a Grade 3 systemic adverse event after afirst dose of the composition. In some aspects, no more than 40% ofsubjects experience a Grade 3 systemic adverse event after a second doseof the composition. In some aspects, administering a composition of theembodiments to human subjects results in no more than 30%, 25%, 20%,15%, 10% or 5% of the subjects experiencing Grade 3 fever, headache,fatigue, chills, myalgia, arthralgia, nausea and/or diarrhea.

TABLE A Intensity Grading* for Solicited Local Adverse Events AE GradeDefinition Pain at 0 Absent Injection 1 Does not interfere with activitySite 2 Interferes with activity and/or repeated use of non-narcotic painreliever >24 hours 3 Prevents daily activity and/or repeated use ofnarcotic pain reliever Redness 0 <2.5 cm 1 2.5-5 cm 2 5.1-10 cm 3 >10 cmSwelling 0 <2.5 cm 1 2.5-5 cm and does not interfere with activity 25.1-10 cm or interferes with activity 3 >10 cm or prevents dailyactivity Itching 0 Absent 1 Mild, no interference with normal activity 2Moderate, some interference with normal activity 3 Significant, preventsnormal activity

TABLE B Intensity Grading* for Solicited Systemic Adverse Events AdverseEvent Grade Definition Fever 0 <38° C. 1 ≥38.0-38.4° C. 2 ≥38.5-38.9° C.3 ≥39° C. Headache 0 Absent 1 Mild, no interference with normal activity2 Moderate, some interference with normal activity and/or repeated useof non- narcotic pain reliever >24 hours 3 Significant; any use ofnarcotic pain reliever and/or prevents daily activity Fatigue 0 Absent 1Mild, no interference with normal activity 2 Moderate, some interferencewith normal activity 3 Significant, prevents normal activity Chills 0Absent 1 Mild, no interference with normal activity 2 Moderate, someinterference with normal activity 3 Significant, prevents normalactivity Myalgia 0 Absent 1 Mild, no interference with normal activity 2Moderate, some interference with normal activity 3 Significant, preventsnormal activity Arthralgia 0 Absent 1 Mild, no interference with normalactivity 2 Moderate, some interference with normal activity 3Significant, prevents normal activity Nausea/ 0 Absent Vomiting 1 Mild,no interference with activity and/or 1-2 episodes/ 24 hours 2 Moderate,some interference with activity and/or >2 episodes/24 hours 3Significant, prevents daily activity, requires outpatient IV hydrationDiarrhea 0 Absent 1 2-3 loose stools over 24 hours 2 4-5 stools over 24hours 3 6 or more watery stools over 24 hours or requires outpatient IVhydration *FDA toxicity grading scale (US Department of Health and HumanServices. Food and Drug Administration (FDA). Guidance for Industry.Toxicity Grading Scale for Healthy Adult and Adolescent VolunteersEnrolled in Preventive Vaccine Clinical Trials. 2007. On the world wideweb atfda.gov/downloads/BiologicsBloodVaccines/GuidanceComplianceRegulatoryInformation/Guidances/Vaccines/ucm091977.pdf;Accessed at: March 2019, incorporated herein by reference); IV =Intravenous.

According to a further aspect, the present invention also provides amethod for expression of at least one polypeptide comprising at leastone peptide or protein derived from a coronavirus, or a fragment orvariant thereof, wherein the method preferably comprises the followingsteps:

-   a) providing at least one nucleic acid of the first aspect or at    least one composition of the second aspect; and-   b) applying or administering said nucleic acid or composition to an    expression system (cells), a tissue, an organism. A suitable cell    for expressing a polypeptide (that is provided by the nucleic acid    of the invention) may be a Drosophila S2 insect cell line.

The method for expression may be applied for laboratory, for research,for diagnostic, for commercial production of peptides or proteins and/orfor therapeutic purposes. The method may furthermore be carried out inthe context of the treatment of a specific disease, particularly in thetreatment of infectious diseases, particularly coronavirus infections,preferably SARS-CoV-2 coronavirus infections and the disease COVID-19.

Likewise, according to another aspect, the present invention alsoprovides the use of the nucleic acid, the composition, the polypeptide,the vaccine, or the kit or kit of parts preferably for diagnostic ortherapeutic purposes, e.g. for expression of an encoded coronavirusantigenic peptide or protein.

In specific embodiments, applying or administering said nucleic acid,polypeptide, composition, vaccine, combination to a tissue or anorganism may be followed by e.g. a step of obtaining induced coronavirusantibodies e.g. SARS-CoV-2 coronavirus specific (monoclonal) antibodiesor a step of obtaining generated SARS-CoV-2 coronavirus proteinconstructs (S protein).

The use may be applied for a (diagnostic) laboratory, for research, fordiagnostics, for commercial production of peptides, proteins, orSARS-CoV-2 coronavirus antibodies and/or for therapeutic purposes. Theuse may be carried out in vitro, in vivo or ex vivo. The use mayfurthermore be carried out in the context of the treatment of a specificdisease, particularly in the treatment of a coronavirus infection (e.g.COVID-19) or a related disorder.

According to a further aspect, the present invention also provides amethod of manufacturing a composition or a vaccine, comprising the stepsof:

-   a) RNA in vitro transcription step using a DNA template in the    presence of a cap analogue to obtain capped mRNA, preferably having    a nucleic acid sequence as provided in Table 3a and 3b;-   b) Purifying the obtained capped RNA of step a) using RP-HPLC,    and/or TFF, and/or Oligo(dT) purification and/or AEX, preferably    using RP-HPLC;-   c) Providing a first liquid composition comprising the purified    capped RNA of step b);-   d) Providing a second liquid composition comprising at least one    cationic lipid as defined herein, a neutral lipid as defined herein,    a steroid or steroid analogue as defined herein, and a PEG-lipid as    defined herein;-   e) Introducing the first liquid composition and the second liquid    composition into at least one mixing means to allow the formation of    LNPs comprising capped RNA;-   f) Purifying the obtained LNPs comprising capped RNA;-   g) optionally, lyophilizing the purified LNPs comprising capped RNA.

Preferably, the mixing means of step e) is a T-piece connector or amicrofluidic mixing device. Preferably, the purifying step f) comprisesat least one step selected from precipitation step, dialysis step,filtration step, TFF step. Optionally, an enzymatic polyadenylation stepmay be performed after step a) or b). Optionally, further purificationsteps may be implemented to e.g. remove residual DNA, buffers, small RNAby-products etc. Optionally, RNA in vitro transcription is performed inthe absence of a cap analog, and an enzymatic capping step is performedafter RNA vitro transcription. Optionally, RNA in vitro transcription isperformed in the presence of at least one modified nucleotide as definedherein.

In embodiments, step a, preferably steps a-c, more preferably all stepsoutlined above (a-g) are performed in an automated device for RNA invitro transcription. Such a device may also be used to produce thecomposition or the vaccine (see aspects 2 and 3). Preferably, a deviceas described in WO2020/002598, in particular, a device as described inclaims 1 to 59 and/or 68 to 76 of WO2020/002598 (and FIGS. 1-18) maysuitably be used.

List of Preferred Embodiments/Items

In the following, particularly preferred embodiments (items 1-275) ofthe invention are provided.

Item List:

-   Item 1. A nucleic acid comprising at least one coding sequence    encoding at least one antigenic peptide or protein that is from or    is derived from a SARS-CoV-2 coronavirus, or an immunogenic fragment    or immunogenic variant thereof, wherein the nucleic acid comprises    at least one heterologous untranslated region (UTR).-   Item 2. Nucleic acid of Item 1, wherein the nucleic acid is suitable    for a vaccine.-   Item 3. Nucleic acid of Item 1 or 2, wherein the at least one    antigenic peptide or protein comprises or consists of at least one    peptide or protein that is or is derived from a structural protein,    an accessory protein, or a replicase protein, or an immunogenic    fragment or immunogenic variant of any of these.-   Item 4. Nucleic acid of Item 3, wherein the structural protein is or    is derived from a spike protein (S), an envelope protein (E), a    membrane protein (M) or a nucleocapsid protein (N), or an    immunogenic fragment or immunogenic variant of any of these.-   Item 5. Nucleic acid of any one of Items 1 to 4, wherein the at    least one antigenic peptide or protein is or is derived from a spike    protein (S), or an immunogenic fragment or immunogenic variant    thereof.-   Item 6. Nucleic acid of any of the preceding Items, wherein the at    least one antigenic peptide or protein comprises or consists of at    least one of the amino acid sequences being identical or at least    70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,    96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 1-111,    274-11663, 13176-13510, 13521-14123, 22732-22758, 22917, 22923,    22929-22964, 26938, 26939 or an immunogenic fragment or immunogenic    variant of any of these.-   Item 7. Nucleic acid of any one of Items 4 to 6, wherein the spike    protein (S) comprises or consists of spike protein fragment S1, or    an immunogenic fragment or immunogenic variant thereof.-   Item 8. Nucleic acid of any one of the preceding Items, wherein the    at least one antigenic peptide or protein comprises or consists of    at least one of the amino acid sequences being identical or at least    70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,    96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 1-27, 29,    31-48, 58-111, 274-1345, 1480-1546, 1614-11663, 13377-13510,    13521-14123, 22732, 22737-22758, 22929-22964 or an immunogenic    fragment or immunogenic variant of any of these.-   Item 9. Nucleic acid of any one of the preceding Items, wherein the    at least one antigenic peptide or protein comprises or consists of    at least one of the amino acid sequences being identical or at least    70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,    96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 27,    1279-1345, 29, 1480-1546, 13243-13309, 22733-22736, 26938, 26939 or    an immunogenic fragment or immunogenic variant of any of these.-   Item 10. Nucleic acid of any one of Items 4 to 9, wherein the spike    protein (S) comprises or consists of a spike protein fragment S1 or    an immunogenic fragment or immunogenic variant thereof, and spike    protein fragment S2 or an immunogenic fragment or immunogenic    variant thereof.-   Item 11. Nucleic acid of any one of the preceding Items, wherein the    at least one antigenic peptide or protein comprises or consists of    at least one of the amino acid sequences being identical or at least    70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,    96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 1-26,    31-48, 58-111, 274-1278, 1614-11663, 13377-13510, 13521-14177,    22732, 22737-22758, 22929-22964 or an immunogenic fragment or    immunogenic variant of any of these.-   Item 12. Nucleic acid of any one of Items 4 to 11, wherein the spike    protein (S) is a pre-fusion stabilized spike protein (S_stab)    comprising at least one pre-fusion stabilizing mutation.-   Item 13. Nucleic acid of Item 12, wherein the at least one    pre-fusion stabilizing mutation comprises the following amino acid    substitutions: K986P and V987P.-   Item 14. Nucleic acid of Item 12 or 13, wherein the at least one    pre-fusion stabilizing mutation comprises a cavity filling mutation.-   Item 15. Nucleic acid of Item 14, wherein the at least one cavity    filling mutation is selected from the list comprising T887W; A1020W;    T887W and A1020W; or P1069F.-   Item 16. Nucleic acid of any one of Items 12 to 15, wherein the at    least one pre-fusion stabilizing mutation comprises a mutated    protonation site.-   Item 17. Nucleic acid of Item 16, wherein the at least one mutated    protonation site is selected from the list comprising H1048Q and    H1064N; H1083N and H1101N; or H1048Q and H1064N and H1083N and    H1101N.-   Item 18. Nucleic acid of any one of Items 12 to 17, wherein the at    least one pre-fusion stabilizing mutation generates at least one    artificial intramolecular disulfide bond.-   Item 19. Nucleic acid of Item 18, wherein the at least one    artificial intramolecular disulfide bond is generated by the    following amino acid substitutions: 17120 and T1077C; 17140 and    Y1110C; P715C and P1069C; G889C and L1034C; 19090 and Y1047C; Q965C    and S1003C; F970C and G999C; A972C and R995C; A890C and V1040C;    T874C and S1055C, or N914C and S1123C.-   Item 20. Nucleic acid of any one of the preceding Items, wherein the    at least one antigenic peptide or protein comprises or consists of    at least one of the amino acid sequences being identical or at least    70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,    96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 10-26,    40-48, 85-111, 341-1278, 1681-2618, 2686-3623, 3691-4628, 4696-5633,    5701-6638, 6706-7643, 7711-8648, 8716-9653, 9721-10658, 10726-11663,    13377-13510, 13521-14123, 22732, 22738, 22740, 22742, 22744, 22746,    22748, 22750, 22752, 22754, 22756, 22758, 22947-22964 or an    immunogenic fragment or immunogenic variant of any of these.-   Item 21. Nucleic acid of any one of the preceding Items, wherein the    at least one antigenic peptide or protein comprises or consists of    at least one of the amino acid sequences being identical or at least    70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,    96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 10-26,    341-407, 609-1278, 13521-13587, 22738, 22740, 22742, 22744, 22746,    22748, 22750, 22752, 22754, 22756, 22758, 22947-22964 or an    immunogenic fragment or immunogenic variant of any of these.-   Item 22. Nucleic acid of any one of the preceding Items, wherein the    at least one coding sequence additionally encodes one or more    heterologous peptide or protein elements selected from a signal    peptide, a linker, a helper epitope, an antigen clustering element,    a trimerization element, a transmembrane element, and/or a    VLP-forming sequence.-   Item 23. Nucleic acid of Item 22, wherein the at least one    heterologous peptide or protein element is a heterologous    antigen-clustering element, a heterologous trimerization element,    and/or a VLP-forming sequence.-   Item 24. Nucleic acid of Item 22 or 23, wherein the at least one    heterologous antigen clustering element is selected from a ferritin    element, a lumazine synthase element, a surface antigen of Hepatitis    B virus (HBsAg), or encapsulin.-   Item 25. Nucleic acid of any one of the preceding Items, wherein the    at least one antigenic peptide or protein comprises or consists of    at least one of the amino acid sequences being identical or at least    70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,    96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 58-75,    85-102, 3624-5633, 7644-9653, 13588-13721, 13856-13989, 22733,    22735, 22736 or an immunogenic fragment or immunogenic variant of    any of these.-   Item 26. Nucleic acid of Item 22 or 23, wherein the at least one    heterologous trimerization element is a foldon element, preferably a    fibritin foldon element.-   Item 27. Nucleic acid of any one of the preceding Items, wherein the    at least one antigenic peptide or protein comprises or consists of    at least one of the amino acid sequences being identical or at least    70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,    96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 76-84,    103-111, 5634-6638, 9654-10658, 13722-13788, 13990-14056, 22734,    26938, 26939 or an immunogenic fragment or immunogenic variant of    any of these.-   Item 28. Nucleic acid of Item 22 or 23, wherein the at least one    VLP-forming sequence is a Woodchuck hepatitis core antigen element    (WhcAg).-   Item 29. Nucleic acid of any one of the preceding Items, wherein the    at least one antigenic peptide or protein comprises or consists of    at least one of the amino acid sequences being identical or at least    70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,    96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 6639-7643,    10659-11663, 13789-13855, 14057-14123 or an immunogenic fragment or    immunogenic variant of any of these.-   Item 30. Nucleic acid of any one of the preceding Items, wherein the    at least one antigenic peptide or protein comprises or consists of    at least one of the amino acid sequences being identical or at least    70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,    96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 1, 10, 21,    22, 25, 27, 274, 341, 408, 475, 542, 743, 810, 1011, 1145, 1212,    1279, 8716, 10726, 22732-22758, 22929-22942, 22947-22964 or an    immunogenic fragment or immunogenic variant of any of these.-   Item 31. Nucleic acid of any one of the preceding Items, wherein the    at least one antigenic peptide or protein comprises or consists of    at least one of the amino acid sequences being identical or at least    70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,    96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 10, 22960,    22961 or 22963 or an immunogenic fragment or immunogenic variant of    any of these.-   Item 32. Nucleic acid of any one of the preceding Items, wherein the    at least one coding sequence comprises or consists of at least one    nucleic acid sequence being identical or at least 70%, 80%, 85%,    86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or    99% identical to any one of SEQ ID NOs: 116-132, 134-138, 140-143,    145-175, 11664-11813, 11815, 11817-12050, 12052, 12054-13147, 13514,    13515, 13519, 13520, 14124-14177, 22759, 22764-22786, 22791-22813,    22818-22839, 22969-23184, 23189-23404, 23409-23624, 23629-23844,    23849-24064, 24069-24284, 24289-24504, 24509-24724, 24729-24944,    24949-25164, 25169-25384, 25389-25604, 25609-25824, 25829-26044,    26049-26264, 26269-26484, 26489-26704, 26709-26937 or a fragment or    variant of any of these sequences.-   Item 33. Nucleic acid of any one of the preceding Items, wherein the    at least one antigenic peptide or protein is an S protein comprising    a pre-fusion stabilizing K986P and V987P mutation comprising or    consisting of the amino acid sequence being identical to SEQ ID NO:    10, or an immunogenic fragment or immunogenic variant of any of    these.-   Item 34. Nucleic acid of any one of the preceding Items, wherein the    at least one coding sequence is a codon modified coding sequence,    wherein the amino acid sequence encoded by the at least one codon    modified coding sequence is preferably not being modified compared    to the amino acid sequence encoded by the corresponding wild type or    reference coding sequence.-   Item 35. Nucleic acid of Item 34, wherein the at least one codon    modified coding sequence is selected from C maximized coding    sequence, CAI maximized coding sequence, human codon usage adapted    coding sequence, G/C content modified coding sequence, and G/C    optimized coding sequence, or any combination thereof.-   Item 36. Nucleic acid of Item 34 or 35, wherein the at least one    codon modified coding sequence is a G/C optimized coding sequence, a    human codon usage adapted coding sequence, or a G/C content modified    coding sequence.-   Item 37. Nucleic acid of any one of the preceding Items, wherein the    at least one coding sequence comprises or consists of a G/C    optimized coding sequence comprising a nucleic acid sequence being    identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,    92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one SEQ    ID NOs: 136-138, 140, 141, 148, 149, 152, 155, 156, 159, 162, 163,    166, 169, 170, 173, 11731-11813, 11815, 11817-11966, 12271-12472,    12743-12944, 13514, 13515, 14124-14132, 14142-14150, 14160-14168,    22759, 22764-22786, 22791-22813, 22818-22839, 22969-23040,    23077-23148, 23189-23260, 23297-23368, 23409-23480, 23517-23588,    23629-23700, 23737-23808, 23849-23920, 23957-24028, 24069-24140,    24177-24248, 24289-24360, 24397-24468, 24509-24580, 24617-24688,    24729-24800, 24837-24908, 24949-25020, 25057-25128, 25169-25240,    25277-25348, 25389-25460, 25497-25568, 25609-25680, 25717-25788,    25829-25900, 25937-26008, 26049-26120, 26157-26228, 26269-26340,    26377-26448, 26489-26560, 26597-26668, 26709-26780, 26817-26888,    26925-26937 or a fragment or variant of any of these sequences.-   Item 38. Nucleic acid of any one of the preceding Items, wherein the    at least one coding sequence comprises or consists of a human codon    usage adapted coding sequence comprising a nucleic acid sequence    being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%,    91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one    of SEQ ID NOs: 142, 143, 145, 150, 153, 157, 160, 164, 167, 171,    174, 11967-12033, 12473-12539, 12945-13011 or a fragment or variant    of any of these sequences.-   Item 39. Nucleic acid of any one of the preceding Items, wherein the    at least one coding sequence comprises or consists of a G/C content    modified coding sequence comprising a nucleic acid sequence being    identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,    92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one SEQ    ID NOs: 146, 147, 151, 154, 158, 161, 165, 168, 172, 175,    12034-12050, 12052, 12054-12203, 12540-12675, 13012-13147, 13519,    13520, 14133-14141, 14151-14159, 14169-14177, 23041-23076,    23149-23184, 23261-23296, 23369-23404, 23481-23516, 23589-23624,    23701-23736, 23809-23844, 23921-23956, 24029-24064, 24141-24176,    24249-24284, 24361-24396, 24469-24504, 24581-24616, 24689-24724,    24801-24836, 24909-24944, 25021-25056, 25129-25164, 25241-25276,    25349-25384, 25461-25496, 25569-25604, 25681-25716, 25789-25824,    25901-25936, 26009-26044, 26121-26156, 26229-26264, 26341-26376,    26449-26484, 26561-26596, 26669-26704, 26781-26816, 26889-26924 or a    fragment or variant of any of these sequences.-   Item 40. Nucleic acid of any one of the preceding Items, wherein the    at least one coding sequence has a G/C content of at least about    50%, 55%, or 60%, preferably of about 63.9%.-   Item 41. Nucleic acid of any one of the preceding Items, wherein the    at least one coding sequence encodes an S protein comprising a    pre-fusion stabilizing K986P and V987P mutation, wherein the coding    sequence comprises or consists of a G/C optimized coding sequence    comprising a nucleic acid sequence being identical to SEQ ID NOs:    137, 23090, 23091, 23093, 23094 or a fragment or variant thereof.-   Item 42. Nucleic acid of any one of the preceding Items, wherein the    at least one heterologous untranslated region is selected from at    least one heterologous 5′-UTR and/or at least one heterologous    3′-UTR.-   Item 43. Nucleic acid of Item 42, wherein the at least one    heterologous 3′-UTR comprises or consists of a nucleic acid sequence    derived from a 3′-UTR of a gene selected from PSMB3, ALB7,    alpha-globin, CASP1, COX6B1, GNAS, NDUFA1 and RPS9, or from a    homolog, a fragment or a variant of any one of these genes.-   Item 44. Nucleic acid of Item 42, wherein the at least one    heterologous 5′-UTR comprises or consists of a nucleic acid sequence    derived from a 5′-UTR of a gene selected from HSD17B4, RPL32, ASAH1,    ATP5A1, MP68, NDUFA4, NOSIP, RPL31, SLC7A3, TUBB4B and UBQLN2, or    from a homolog, a fragment or variant of any one of these genes.-   Item 45. Nucleic acid of Item 42, wherein the at least one    heterologous 5′-UTR and the at least one heterologous 3′ UTR is    selected from UTR design a-1 (HSD17B4/PSMB3), a-3 (SLC7A3/PSMB3),    e-2 (RPL31/RPS9), and i-3 (−/muag), wherein UTR design a-1    (HSD17B4/PSMB3) and i-3 (−/muag) are particularly preferred.-   Item 46. Nucleic acid of any one of the preceding Items, wherein the    nucleic acid comprises at least one poly(A) sequence, preferably    comprising 30 to 200 adenosine nucleotides and/or at least one    poly(C) sequence, preferably comprising 10 to 40 cytosine    nucleotides.-   Item 47. Nucleic acid of any one of the preceding Items, wherein the    nucleic acid comprises at least one histone stem-loop.-   Item 48. Nucleic acid of any one of the preceding Items, wherein the    nucleic acid is a DNA or an RNA.-   Item 49. Nucleic acid of any one of the preceding Items, wherein the    nucleic acid is a coding RNA.-   Item 50. Nucleic acid of Item 49, wherein the coding RNA is an mRNA,    a self-replicating RNA, a circular RNA, or a replicon RNA.-   Item 51. Nucleic acid of any one of the preceding Items, wherein the    nucleic acid, preferably the coding RNA, is an mRNA.-   Item 52. Nucleic acid of Item 51, wherein the mRNA is not a replicon    RNA or a self-replicating RNA.-   Item 53. Nucleic acid of Item 51, wherein the mRNA comprises at    least one poly(A) sequence comprising 30 to 200 adenosine    nucleotides and the 3′ terminal nucleotide is an adenosine.-   Item 54. Nucleic acid of any one of Items 48 to 51, wherein the RNA,    preferably the coding RNA, comprises a 5′-cap structure, preferably    m7G, cap0, cap1, cap2, a modified cap0 or a modified cap1 structure,    preferably a 5′-cap1 structure.-   Item 55. Nucleic acid of any one of Items 48 to 54, wherein the    nucleic acid, preferably the mRNA, comprises the following elements    in 5′- to 3′-direction:    -   A) 5′-cap1 structure;    -   B) coding sequence according to SEQ ID NO. 137, or a fragment or        variant thereof;    -   C) 3′-UTR derived from a 3′-UTR of an alpha-globin gene,        preferably according to SEQ ID NO: 267 or 268;    -   D) poly(A) sequence comprising about 64 A nucleotides;    -   E) poly(C) sequence comprising about 30 C nucleotides;    -   F) histone stem-loop according to SEQ ID NOs: 178 or 179.-   Item 56. Nucleic acid of any one of Items 48 to 54, wherein the    nucleic acid, preferably the mRNA, comprises the following elements    in 5′- to 3′-direction:    -   A) 5′-cap1 structure;    -   B) 5′-UTR derived from a 5′-UTR of a HSD17B4 gene, preferably        according to SEQ ID NO: 231 or 232; C) coding sequence according        to SEQ ID NO. 137, or a fragment or variant thereof;    -   D) 3′-UTR derived from a 3′-UTR of a PSMB3 gene, preferably        according to SEQ ID NO: 253 or 254;    -   E) a histone stem-loop selected from SEQ ID NOs: 178 or 179;    -   F) poly(A) sequence comprising about 100 A nucleotides.-   Item 57. Nucleic acid of Item 56, wherein the 3′ terminal nucleotide    is an adenosine.-   Item 58. Nucleic acid of any one of Items 48 to 54, wherein the    nucleic acid, preferably the mRNA, comprises the following elements    in 5′- to 3′-direction:    -   A) 5′-cap1 structure;    -   B) coding sequence according to SEQ ID NO. 23090 or 23091, or a        fragment or variant thereof;    -   C) 3′-UTR derived from a 3′-UTR of an alpha-globin gene,        preferably according to SEQ ID NO: 267 or 268;    -   D) poly(A) sequence comprising about 64 A nucleotides;    -   E) poly(C) sequence comprising about 30 C nucleotides;-   F) histone stem-loop according to SEQ ID NOs: 178 or 179.-   Item 59. Nucleic acid of any one of Items 48 to 54, wherein the    nucleic acid, preferably the mRNA, comprises the following elements    in 5′- to 3′-direction:    -   A) 5′-cap1 structure;    -   B) 5′-UTR derived from a 5′-UTR of a HSD17B4 gene, preferably        according to SEQ ID NO: 231 or 232;    -   C) coding sequence according to SEQ ID NO. 23090 or 23091, or a        fragment or variant thereof;    -   D) 3′-UTR derived from a 3′-UTR of a PSMB3 gene, preferably        according to SEQ ID NO: 253 or 254;    -   E) a histone stem-loop selected from SEQ ID NOs: 178 or 179;    -   F) poly(A) sequence comprising about 100 A nucleotides.-   Item 60. Nucleic acid of any one of the preceding Items, wherein the    nucleic acid comprises or consists of a nucleic acid sequence,    preferably an RNA sequence, which is identical or at least 70%, 80%,    85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,    98%, or 99% identical to a nucleic acid sequence selected from the    group consisting of SEQ ID NOs: 148-175, 12204-13147, 14142-14177,    22786-22839, 23189-23404, 23409-23624, 23629-23844, 23849-24064,    24069-24284, 24289-24504, 24509-24724, 24729-24944, 24949-25164,    25169-25384, 25389-25604, 25609-25824, 25829-26044, 26049-26264,    26269-26484, 26489-26704, 26709-26937148 or a fragment or variant of    any of these sequences.-   Item 61. Nucleic acid of any one of the preceding Items, wherein the    nucleic acid comprises or consists of a nucleic acid sequence,    preferably an RNA sequence, which is identical or at least 70%, 80%,    85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,    98%, or 99% identical to a nucleic acid sequence selected from the    group consisting of SEQ ID NOs: 149, 156, 12338, 150, 157, 151, 158,    12541, 163, 170, 12810, 164, 171, 165, 172, 13013, 12342-12351,    12545-12554, 12814-12823, 13017-13026, 14133 ora fragment or variant    of any of these sequences, preferably selected from SEQ ID NOs: 149,    150, 151, 163, 164, 165 or a fragment or variant of any of these    sequences.-   Item 62. Nucleic acid of any one of the preceding Items, wherein the    nucleic acid comprises or consists of a nucleic acid sequence,    preferably an RNA sequence, which is identical or at least 70%, 80%,    85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,    98%, or 99% identical to a nucleic acid sequence selected from SEQ    ID NOs: 163 or a fragment or variant thereof.-   Item 63. Nucleic acid of any one of the preceding Items, wherein the    nucleic acid comprises or consists of a nucleic acid sequence,    preferably an RNA sequence, which is identical or at least 70%, 80%,    85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,    98%, or 99% identical to a nucleic acid sequence selected from SEQ    ID NOs: 149 or a fragment or variant thereof.-   Item 64. Nucleic acid of any one of the preceding Items, wherein the    nucleic acid comprises or consists of a nucleic acid sequence,    preferably an RNA sequence, which is identical or at least 70%, 80%,    85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,    98%, or 99% identical to a nucleic acid sequence selected from SEQ    ID NOs: 24837 or a fragment or variant thereof.-   Item 65. Nucleic acid of any one of the preceding Items, wherein the    nucleic acid comprises or consists of a nucleic acid sequence,    preferably an RNA sequence, which is identical or at least 70%, 80%,    85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,    98%, or 99% identical to a nucleic acid sequence selected from the    group consisting of SEQ ID NOs: 23311, 23531, 24851 or a fragment or    variant thereof.-   Item 66. Nucleic acid of any one of the preceding Items, wherein the    nucleic acid comprises or consists of a nucleic acid sequence,    preferably an RNA sequence, which is identical or at least 70%, 80%,    85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,    98%, or 99% identical to a nucleic acid sequence selected from the    group consisting of SEQ ID NOs: 23310, 23530, 24850 or a fragment or    variant thereof.-   Item 67. Nucleic acid of any one of the preceding Items, wherein the    nucleic acid comprises or consists of a nucleic acid sequence,    preferably an RNA sequence, which is identical or at least 70%, 80%,    85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,    98%, or 99% identical to a nucleic acid sequence selected from the    group consisting of SEQ ID NOs: 23313, 23533, 24853, 23314, 23534,    24854 or a fragment or variant thereof.-   Item 68. Nucleic acid of any one of the preceding Items, wherein the    nucleic acid is an RNA that does not comprise a    1-methylpseudouridine substitution.-   Item 69. Nucleic acid of any one of the preceding Items, wherein the    nucleic acid is an RNA that does not comprise chemically modified    nucleotides.-   Item 70. Nucleic acid of any one of the preceding Items, wherein the    nucleic acid is an in vitro transcribed RNA, wherein RNA in vitro    transcription has been performed in the presence of a sequence    optimized nucleotide mixture and a cap analog, preferably wherein    the sequence optimized nucleotide mixture does not comprise    chemically modified nucleotides.-   Item 71. Nucleic acid of any one of the preceding Items, wherein the    nucleic acid is a purified RNA, preferably an RNA that has been    purified by RP-HPLC and/or TFF.-   Item 72. Nucleic acid of any one of the preceding Items, wherein the    nucleic acid is a purified RNA that has been purified by RP-HPLC    and/or TFF and comprises about 5%, 10%, or 20% less double stranded    RNA side products as an RNA that has not been purified with RP-HPLC    and/or TFF.-   Item 73. Nucleic acid of any one of the preceding Items, wherein the    nucleic acid is a purified RNA that has been purified by RP-HPLC    and/or TFF and comprises about 5%, 10%, or 20% less double stranded    RNA side products as an RNA that has been purified with Oligo dT    purification, precipitation, filtration and/or anion exchange    chromatography.-   Item 74. A composition comprising at least one nucleic acid as    defined in any one of Items 1 to 73, wherein the composition    optionally comprises at least one pharmaceutically acceptable    carrier.-   Item 75. Composition of Items 74, wherein the composition comprises    an mRNA according to SEQ ID NOs: 149, 163, 24837, 23311, 23531,    23310, 23530, 23313 or 23533 or a fragment or variant of any of    these sequences.-   Item 76. Composition of Item 74, wherein the composition is a    multivalent composition comprising a plurality or at least more than    one of the nucleic acid as defined in in any one of Items 1 to 73.-   Item 77. Composition of Item 76, wherein the plurality or at least    more than one of the nucleic acid sequences of the multivalent    composition each encode a different spike protein, preferably a    prefusion stabilized spike protein.-   Item 78. Composition of Item 77, wherein the different spike    proteins or prefusion stabilized spike proteins are derived from    different SARS-CoV-2 virus variants/isolates-   Item 79. Composition of Item 78, wherein the different spike    proteins or prefusion stabilized spike proteins are derived from at    least B.1.1.7, B.1.351, P.1, or CAL.20C.-   Item 80. Composition of Item 78, wherein the different spike    proteins or prefusion stabilized spike proteins have amino acid    changes in the S protein comprising:    -   (i) delH69, delV70, Y453F, D614G, 1692V and M1229I;    -   (ii) delH69, delV70, delY144, N501Y, A570D, D614G, P681H, T716I,        S982A and D1118H;    -   (iii) L18F, D80A, D215G, delL242, delA243, delL244, R2461,        K417N, E484K, N501Y, D614G and A701V;    -   (iv) L18F, T20N, P26S, D138Y, R190S, K417T, E484K, N501Y, D614G,        H655Y and T10271; and/or    -   (v) S13I, W152C, L452R, and D614G.-   Item 81. Composition of any one of Items 76 to 78, wherein the    multivalent composition comprises at least two nucleic acid species    comprising a coding sequence encoding an amino acid sequence being    identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,    92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of    SEQ ID NOs: 10, 22961; 22960, 22963, 22941, 22964.-   Item 82. Composition of any one of Items 76 to 78, wherein the    multivalent composition comprises at least two RNA species being    identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,    92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of    SEQ ID NOs: 149 or 24837, 23531 or 24851, 23530 or 24850, 23533 or    24853, 23439 or 24759 or 23534 or 24854.-   Item 83. Composition of any one of Items 74 to 80, wherein the    composition comprises mRNA with an RNA integrity of 70% or more.-   Item 84. Composition of any one of Items 74 to 83, wherein the    composition comprises mRNA with a capping degree of 70% or more,    preferably wherein at least 70%, 80%, or 90% of the mRNA species    comprise a Cap1 structure.-   Item 85. Composition of any one of Items 74 to 84, wherein the at    least one nucleic acid is complexed or associated with or at least    partially complexed or partially associated with one or more    cationic or polycationic compound, preferably cationic or    polycationic polymer, cationic or polycationic polysaccharide,    cationic or polycationic lipid, cationic or polycationic protein,    cationic or polycationic peptide, or any combinations thereof.-   Item 86. Composition of Item 85, wherein the at least one nucleic    acid is complexed or associated with one or more lipids or    lipid-based carriers, thereby forming liposomes, lipid nanoparticles    (LNP), lipoplexes, and/or nanoliposomes, preferably encapsulating    the at least one nucleic acid.-   Item 87. Composition of Items 85 or 86, wherein the at least one    nucleic acid is complexed with one or more lipids thereby forming    lipid nanoparticles.-   Item 88. Composition of Item 86 or 87, wherein the LNP comprises a    cationic lipid according to formula III-3:

-   Item 89. Composition of any one of Items 86-88, wherein the LNP    comprises a PEG lipid of formula (IVa):

-   -   wherein n has a mean value ranging from 30 to 60, preferably        wherein n has a mean value of about 45, 46, 47, 48, 49, 50, 51,        52, 53, 54, most preferably wherein n has a mean value of 49 or        45.

-   Item 90. Composition of any one of Items 86-88, wherein the LNP    comprises a PEG lipid of formula (IVa):

wherein n is an integer selected such that the average molecular weightof the PEG lipid is about 2500 g/mol.

-   Item 91. Composition of any one of Items 86-90, wherein the LNP    comprises one or more neutral lipids and/or one or more steroid or    steroid analogues.-   Item 92. Composition of Item 91, wherein the neutral lipid is    1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), preferably    wherein the molar ratio of the cationic lipid to DSPC is in the    range from about 2:1 to about 8:1.-   Item 93. Composition of Item 91, wherein the steroid is cholesterol,    preferably wherein the molar ratio of the cationic lipid to    cholesterol is in the range from about 2:1 to about 1:1.-   Item 94. Composition of any one of Items 86-93, wherein the LNP    comprises    -   (i) at least one cationic lipid, preferably a lipid of formula        (III), more preferably lipid 111-3;    -   (ii) at least one neutral lipid, preferably        1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC);    -   (iii) at least one steroid or steroid analogue, preferably        cholesterol; and    -   (iv) at least one polymer conjugated lipid, preferably a        PEG-lipid derived from formula (IVa, with n=49),    -   wherein (i) to (iv) are in a molar ratio of about 20-60%        cationic lipid, 5-25% neutral lipid, 25-55% sterol, and 0.5-15%        PEG-lipid.-   Item 95. Composition of any one of Items 86-93, wherein the LNP    comprises    -   (i) at least one cationic lipid, preferably a lipid of formula        (III), more preferably lipid 111-3;    -   (ii) at least one neutral lipid, preferably        1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC);    -   (iii) at least one steroid or steroid analogue, preferably        cholesterol; and    -   (iv) at least one polymer conjugated lipid, preferably a        PEG-lipid derived from formula (IVa, with n=45),    -   wherein (i) to (iv) are in a molar ratio of about 20-60%        cationic lipid, 5-25% neutral lipid, 25-55% sterol, and 0.5-15%        PEG-lipid.-   Item 96. Composition of Item 94 or 95, wherein (i) to (iv) are in a    molar ratio of about 50:10:38.5:1.5, preferably 47.5:10:40.8:1.7 or    more preferably 47.4:10:40.9:1.7.-   Item 97. Composition of any one of Items 87 to 96, wherein the    nucleic acid is RNA and the composition comprises less than about    20% free (non complexed or non-encapsulated) RNA, preferably less    than about 15% free RNA, more preferably less than about 10% free    RNA.-   Item 98. Composition of any one of Items 87 to 97, wherein the wt/wt    ratio of lipid to nucleic acid is from about 10:1 to about 60:1,    preferably from about 20:1 to about 30:1, for example about 25:1.-   Item 99. Composition of any one of Items 87 to 98, wherein the n/p    ratio of the LNPs encapsulating the nucleic acid is in a range from    about 1 to about 10, preferably in a range from about 5 to about 7,    more preferably about 6.-   Item 100. Composition of any one of Items 87 to 99, wherein the    composition has a polydispersity index (PDI) value of less than    about 0.4, preferably of less than about 0.3, more preferably of    less than about 0.2, most preferably of less than about 0.1.-   Item 101. Composition of any one of Items 86 to 100, wherein the    LNPs have a Z-average size in a range of about 60 nm to about 120    nm, preferably less than about 120 nm, more preferably less than    about 100 nm, most preferably less than about 80 nm.-   Item 102. Composition of any one of Items 86 to 101, wherein the    LNPs comprise less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%,    1% LNPs that have a particle size exceeding about 500 nm.-   Item 103. Composition of any one of Items 86 to 102, wherein the    LNPs comprise less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%,    1% LNPs that have a particle size smaller than about 20 nm.-   Item 104. Composition of any one of Items 86 to 103, wherein at    least about 80%, 85%, 90%, 95% of lipid-based carriers have a    spherical morphology, preferably comprising a solid core or    partially solid core.-   Item 105. Composition of any one of Items 86 to 104, wherein the    composition has a turbidity ranging from about 150 FNU to about 0.0    FNU, preferably of about 50 FNU or less, more preferably of about 25    FNU or less.-   Item 106. Composition of any one of Items 74 to 105, further    comprising a sugar in a concentration of about 50 to about 300 mM,    preferably sucrose in a concentration of about 150 mM.-   Item 107. Composition of any one of Items 74 to 106, further    comprising a salt in a concentration of about 10 mM to about 200 mM,    preferably NaCl in a concentration of about 75 mM.-   Item 108. Composition of any one of Items 74 to 107, further    comprising a buffering agent in a concentration 1 mM to about 100    mM, preferably Na₃PO₄ in a concentration of about 10 mM.-   Item 109. Composition of any one of Items 74 to 108, wherein the    composition has a pH in a range of about pH 7.0 to about pH 8.0,    preferably of about pH 7.4.-   Item 110. Composition of any one of Items 86 to 109, comprising    lipid nanoparticles encapsulating an RNA encoding a SARS-CoV-2 S    protein comprising a pre-fusion stabilizing K986P and V987P mutation    -   wherein the LNPs comprise        -   (i) cationic lipid of formula III-3;        -   (ii) 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC);        -   (iii) cholesterol; and        -   (iv) PEG-lipid of formula IVa (n=49)),    -   wherein (i) to (iv) are in a molar ratio of about 47.4% cationic        lipid, 10% DSPC, 40.9 cholesterol, 1.7% PEG-lipid;    -   wherein the RNA is identical or at least 70%, 80%, 85%, 86%,        87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or        99% identical to a nucleic acid sequence of SEQ ID NO. 163 or        149;    -   wherein the RNA is not chemically modified;    -   wherein the RNA comprises a 5′-Cap1 structure;    -   wherein the integrity of the RNA is at least about 70%;    -   wherein the n/p ratio of the LNPs encapsulating the RNA is about        6;    -   wherein the LNPs encapsulating the RNA have a Z-average size of        about 60 nm to about 120 nm;    -   wherein the composition comprises less than about 20% free (non        complexed; non-encapsulated) RNA;    -   optionally, wherein the composition further comprises sucrose in        a concentration of about 150 mM, NaCl in a concentration of        about 75 mM, Na₃PO₄ in a concentration of about 10 mM;    -   optionally, wherein the composition has a pH of about pH 7.4.-   Item 111. Composition of any one of Items 86 to 109, comprising    lipid nanoparticles encapsulating an RNA encoding a SARS-CoV-2 S    protein comprising a pre-fusion stabilizing K986P and V987P mutation    -   wherein the LNPs comprise        -   (i) cationic lipid of formula III-3;        -   (ii) 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC);        -   (iii) cholesterol; and        -   (iv) PEG-lipid of formula IVa (n=45)),    -   wherein (i) to (iv) are in a molar ratio of about 47.4% cationic        lipid, 10% DSPC, 40.9 cholesterol, 1.7% PEG-lipid;    -   wherein the RNA is identical or at least 70%, 80%, 85%, 86%,        87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or        99% identical to a nucleic acid sequence of SEQ ID NO. 163 or        149;    -   wherein the RNA is not chemically modified;    -   wherein the RNA comprises a 5′-Cap1 structure;    -   wherein the integrity of the RNA is at least about 70%;    -   wherein the n/p ratio of the LNPs encapsulating the RNA is about        6;    -   wherein the LNPs encapsulating the RNA have a Z-average size of        about 60 nm to about 120 nm;    -   wherein the composition comprises less than about 20% free (non        complexed) RNA;    -   optionally, wherein the composition further comprises sucrose in        a concentration of about 150 mM, NaCl in a concentration of        about 75 mM, Na₃PO₄ in a concentration of about 10 mM;    -   optionally, wherein the composition has a pH of about pH 7.4.-   Item 112. Composition of any one of Items 86 to 110, comprising an    RNA that is not chemically modified, which is identical or at least    70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,    96%, 97%, 98%, or 99% identical to a nucleic acid sequence of SEQ ID    NO. 163 formulated in lipid nanoparticles (LNPs), which have a molar    ratio of approximately 50:10:38.5:1.5, preferably 47.5:10:40.8:1.7    or more preferably 47.4:10:40.9:1.7 proportion (mol %) of cationic    lipid III-3, DSPC, cholesterol and PEG-lipid of formula (IVa).-   Item 113. Composition of any one of Items 86 to 110, comprising an    RNA that is not chemically modified, which is identical or at least    70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,    96%, 97%, 98%, or 99% identical to a nucleic acid sequence of SEQ ID    NO. 149 formulated in lipid nanoparticles (LNPs), which have a molar    ratio of approximately 50:10:38.5:1.5, preferably 47.5:10:40.8:1.7    or more preferably 47.4:10:40.9:1.7 proportion (mol %) of cationic    lipid III-3, DSPC, cholesterol and PEG-lipid of formula (IVa).-   Item 114. Composition any one of Items 74 to 112, wherein the    composition comprises a mRNA encoding a SARS-CoV-2 spike protein (S)    that is a pre-fusion stabilized spike protein (S_stab) comprising at    least one pre-fusion stabilizing mutation.-   Item 115. Composition of Item 114, wherein the mRNA encodes a    SARS-CoV-2 spike protein at least 95% identical to SEQ ID NO: 163 or    encodes a coronavirus spike protein identical to SEQ ID NO: 163.-   Item 116. Composition of Item 114, wherein the LNP comprises    -   (i) at least one cationic lipid;    -   (ii) at least one neutral lipid;    -   (iii) at least one steroid or steroid analogue; and    -   (iv) at least one PEG-lipid,    -   wherein (i) to (iv) are in a molar ratio of about 20-60%        cationic lipid, 5-25% neutral lipid, 25-55% sterol, and 0.5-15%        PEG-lipid.-   Item 117. Composition of Item 114, wherein the LNP comprises    -   (i) at least one cationic lipid according to formula III-3;    -   (ii) DSPC;    -   (iii) cholesterol; and    -   (iv) a PEG-lipid, according to formula IVa,    -   wherein (i) to (iv) are in a molar ratio of about 20-60%        cationic lipid, 5-25% neutral lipid, 25-55% sterol, and 0.5-15%        PEG-lipid.-   Item 118. Composition of Item 114, wherein the LNP comprises    -   (i) at least one cationic lipid according to formula III-3;    -   (ii) DSPC;    -   (iii) cholesterol; and    -   (iv) a PEG-lipid, according to formula IVa,    -   wherein (i) to (iv) are in a molar ratio of about        47.5:10:40.8:1.7.-   Item 119. Composition of Item 114, wherein the LNP comprises:    -   (i) at least one cationic lipid according to formula III-3;    -   (ii) DSPC;    -   (iii) cholesterol; and    -   (iv) a PEG-lipid, according to formula IVa,    -   wherein (i) to (iv) are in a molar ratio of 47.4:10:40.9:1.7.-   Item 120. Composition of anyone of Items 107-118, wherein the ratio    of mRNA to total lipid is about 0.03-0.04 w/w.-   Item 121. Composition of Item 120, wherein the mRNA is complexed    with one or more lipids thereby forming lipid nanoparticles (LNP),    wherein the LNP comprises    -   (i) at least one cationic lipid according to formula III-3;    -   (ii) DSPC;    -   (iii) cholesterol; and    -   (iv) a PEG-lipid, according to formula IVa,    -   wherein (i) to (iv) are in a molar ratio of about        47.5:10:40.8:1.7, and    -   wherein the ratio of mRNA to total lipid is about 0.03-0.04 w/w.-   Item 122. Composition of Item 120, wherein the mRNA is complexed    with one or more lipids thereby forming lipid nanoparticles (LNP),    wherein the LNP comprises:    -   (i) at least one cationic lipid according to formula III-3;    -   (ii) DSPC;    -   (iii) cholesterol; and    -   (iv) a PEG-lipid, according to formula IVa,    -   wherein (i) to (iv) are in a molar ratio of 47.4:10:40.9:1.7,        and    -   wherein the ratio of mRNA to total lipid is about 0.03-0.04 w/w.-   Item 123. Composition of any one of Items 74-99 or 110-122, wherein    the composition is a lyophilized composition.-   Item 124. Composition of Item 123, wherein the lyophilized    composition has a water content of less than about 10%.-   Item 125. Composition of Item 124, wherein the lyophilized    composition has a water content of between about 0.5% and 5%.-   Item 126. Composition any one of Items 86 to 122, wherein the    nucleic acid is RNA and wherein the composition is stable for at    least about two weeks after storage as a liquid at temperatures of    about 5° C.-   Item 127. Composition of Item 126, wherein the nucleic acid is RNA    and wherein the composition is stable for at least 1 month after    storage as a liquid at temperatures of about 5° C.-   Item 128. Composition of Item 126, wherein the nucleic acid is RNA    and wherein the composition is stable for about 2 weeks to about 1    month, 2 months, 3 months, 4 months, 5 months, 6 months or 1 year    after storage as a liquid at temperatures of about 5° C.-   Item 129. Composition of Item 126, wherein the nucleic acid is RNA    and wherein at least 70%, 75%, 80%, 85%, 90% or 95% of the RNA is    intact at least about two weeks after storage as a liquid at    temperatures of about 5° C.-   Item 130. Composition of Item 129, wherein the nucleic acid is RNA    and wherein at least 70%, 75%, 80%, 85%, 90% or 95% of the RNA is    intact at least 1 month after storage as a liquid at temperatures of    about 5° C.-   Item 131. Composition of Item 126, wherein the nucleic acid is RNA    and wherein at least 70%, 75%, 80%, 85%, 90% or 95% of the RNA is    intact about 2 weeks to about 1 month, 2 months, 3 months, 4 months,    5 months, 6 months or 1 year after storage as a liquid at    temperatures of about 5° C.-   Item 132. Composition of Item 126, wherein the nucleic acid is RNA    and wherein at least 80% of the RNA is intact after about two weeks    of storage as a liquid at temperatures of about 5° C.-   Item 133. Composition any one of Items 86 to 132, wherein the    composition comprises an aggregation reducing lipid.-   Item 134. Composition any one of Items 86 to 133, wherein the    nucleic acid is RNA and wherein the concentration of the RNA is in a    range from about 10 μg/ml to about 10 mg/ml, preferably in a range    from about 100 μg/ml to about 1 mg/ml.-   Item 135. Composition any one of Items 86 to 133, wherein the    nucleic acid is RNA and wherein the concentration of the RNA is at    least 100 μg/ml, more preferably at least 200 μg/ml, most preferably    at least 500 μg/ml.-   Item 136. Composition any one of Items 86 to 135, wherein the    nucleic acid is RNA and wherein the RNA has an RNA integrity of at    least about 50%, preferably of at least about 60%, more preferably    of at least about 70%, most preferably of at least about 80%.-   Item 137. Composition any one of Items 86 to 136, wherein the    nucleic acid is RNA and wherein the composition comprises less than    about 20% free RNA, preferably less than about 15% free RNA, more    preferably less than about 10% free RNA.-   Item 138. Composition any one of Items 86 to 137, wherein the    nucleic acid is RNA and wherein the composition comprises less than    about 100 nM divalent cations per g RNA, preferably less than about    50 nM divalent cations per g RNA, more preferably less than about 10    nM divalent cations per g RNA.-   Item 139. Composition of Item 138, wherein the divalent cations are    selected from Mg2+ and/or Ca2+.-   Item 140. Composition of any one of Items 86 to 139, wherein the    concentration of lipid is in a range from about 250 μg/ml to about    250 mg/ml, preferably in a range from about 2.5 mg/ml to about 25    mg/ml.-   Item 141. Composition of any one of Items 86 to 140, wherein the    concentration of lipid is at least about 2.5 mg/ml, preferably at    least 5 mg/ml, more preferably at least 12.5 mg/ml.-   Item 142. Composition of any one of Items 133 to 142, wherein the    concentration of aggregation reducing lipid is in a range from about    17.5 μg/ml to about 17.5 mg/ml, preferably in a range from about 175    μg/ml to about 1.75 mg/ml.-   Item 143. Composition of any one of Items 133 to 142, wherein the    concentration of aggregation reducing lipid is at least about 175    μg/ml, preferably at least about 350 μg/ml, more preferably at least    875 μg/ml.-   Item 144. Composition of any one of Items 86 to 143, wherein the    nucleic acid is RNA and wherein the wt/wt ratio of lipid to the RNA    is from about 10:1 to about 60:1, preferably from about 20:1 to    about 30:1, more preferably about 25:1.-   Item 145. Composition of any one of Items 86 to 144, wherein the    nucleic acid is RNA and wherein the N/P ratio of the lipid-based    carriers to the RNA is in a range from about 1 to about 10,    preferably in a range from about 5 to about 7, more preferably about    6.-   Item 146. Composition of any one of Item 86 to 143, wherein the    nucleic acid is RNA and wherein the lipid-based carriers    encapsulating the RNA comprise an aggregation reducing lipid in a    molar ratio of about 0.5%-15%, preferably in a molar ratio of about    1.0% to about 2.5%, more preferably in a molar ratio of about 1.7%.-   Item 147. Composition of any one of Item 133 to 143, wherein the    aggregation reducing lipid is a polymer conjugated lipid, e.g. a    PEG-conjugated lipid.-   Item 148. Composition of any one of Item 86 to 147, wherein the    nucleic acid is RNA and wherein the RNA and lipid-based carrier    encapsulating the RNA have been purified by at least one    purification step, preferably by at least one step of TFF and/or at    least one step of clarification and/or at least one step of    filtration.-   Item 149. Composition of any one of Item 86 to 148, wherein the    composition comprises less than about 500 ppM ethanol, preferably    less than about 50 ppM ethanol, more preferably less than about 5    ppM ethanol.-   Item 150. Composition of any one of Item 86 to 154, wherein the    composition has an osmolarity of about 250 mOsmol/kg to about 450    mOsmol/kg, preferably of about 335 mOsmol/kg.-   Item 151. Composition of any one of Item 86 to 150, wherein the    composition is stable for at least 1 week, preferably for at least 2    weeks, more preferably for at least 3 weeks, most preferably for at    least 4 weeks after storage as a liquid at about 25° C.-   Item 152. Composition of any one of Item 86 to 151, wherein the    composition is stable for at least 1 day, preferably for at least 2    days, more preferably for at least 3 days, most preferably for at    least 4 days after storage as a liquid at about 40° C.-   Item 153. Composition of any one of Item 86 to 152, wherein upon    storage as a liquid, the integrity of the RNA decreases less than    about 30%, preferably less than about 20%, more preferably less than    about 10%.-   Item 154. Composition of any one of Item 86 to 153, wherein upon    storage as a liquid, the amount of free RNA does not increase by    more than 10%, preferably by not more than 5%.-   Item 155. Composition of any one of Item 86 to 154, wherein the    nucleic acid is RNA and wherein upon storage as a liquid, the PDI    value of the lipid-based carriers encapsulating the RNA does not    increase by more than a value of about 0.2, preferably by not more    than a value of about 0.1.-   Item 156. Composition of any one of Item 86 to 155, wherein the    nucleic acid is RNA and wherein upon storage as a liquid, the    Z-average size of the lipid-based carriers encapsulating the RNA    does not increase by more than 20%, preferably by not more than 10%.-   Item 157. Composition of any one of Item 86 to 156, wherein upon    storage as a liquid, the turbidity of the composition does not    increase by more than 20%, preferably by not more than 10%.-   Item 158. Composition of any one of Item 86 to 157, wherein upon    storage as a liquid, the pH and/or the osmolality does not increase    or decrease by more than 20%, preferably by not more than 10%.-   Item 159. Composition of any one of Item 86 to 158, wherein upon    storage as a liquid, the potency of the composition decreases less    than about 30%, preferably less than about 20%, more preferably less    than about 10%.-   Item 160. Composition of any one of Items 86 to 133, wherein the    nucleic acid is RNA and wherein the RNA is a purified RNA,    preferably an RP-HPLC purified RNA and/or a tangential flow    filtration (TFF) purified RNA.-   Item 161. Composition of any one of Items 74 to 160, additionally    comprising at least one antagonist of at least one RNA sensing    pattern recognition receptor, preferably at least one antagonist of    a TLR7 receptor and/or a TLR8 receptor.-   Item 162. Composition of Item 161, wherein the at least one    antagonist of a TLR7 receptor and/or a TLR8 receptor is a single    stranded oligonucleotide, preferably p5′-GAG CGmG CCA-3′.-   Item 163. A polypeptide for a vaccine comprising at least one    antigenic peptide or protein that is or is derived from a    coronavirus SARS-CoV-2, or an immunogenic fragment or immunogenic    variant thereof, preferably wherein the amino acid sequences of said    antigenic peptide or protein is identical or at least 70%, 80%, 85%,    86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or    99% identical to any one of amino acid sequences SEQ ID NOs: 1-111,    274-11663, 13176-13510, 13521-14123, 22732-22758, 22917, 22923,    22929-22964, 26938, 26939, or an immunogenic fragment or immunogenic    variant of any of these.-   Item 164. A vaccine comprising the nucleic acid of any one of Items    1 to 73, and/or the composition of any one of Items 74 to 162,    and/or the polypeptide of Item 163.-   Item 165. A vaccine of Item 164, wherein the vaccine elicits an    adaptive immune response, preferably a protective adaptive immune    response against a coronavirus, preferably against coronavirus    SARS-CoV-2.-   Item 166. A vaccine of Item 164 or 165, wherein the vaccine is a    multivalent vaccine comprising a plurality or at least more than one    of the nucleic acid as defined in any one of Items 1 to 73, or a    plurality or at least more than one of the compositions as defined    in any one of Items 74 to 162.-   Item 167. A Kit or kit of parts, comprising the nucleic acid of any    one of Items 1 to 73, and/or the composition of any one of Items 74    to 162, and/or the polypeptide of Item 163, and/or the vaccine of    Item 164 to 166, optionally comprising a liquid vehicle for    solubilising, and, optionally, technical instructions providing    information on administration and dosage of the components.-   Item 168. Nucleic acid of any one of Items 1 to 73, the composition    of any one of Items 74 to 162 the polypeptide of-   Item 163, the vaccine of Item 164 to 166, the kit or kit of parts of    Item 167, for use as a medicament.-   Item 169. Nucleic acid of any one of Items 1 to 73, the composition    of any one of Items 74 to 162, the polypeptide of-   Item 163, the vaccine of Item 164 to 166, the kit or kit of parts of    Item 167, for use in the treatment or prophylaxis of an infection    with a coronavirus, preferably a SARS-CoV-2 coronavirus, or of a    disorder related to such an infection, preferably COVID-19.-   Item 170. A method of treating or preventing a disorder, wherein the    method comprises applying or administering to a subject in need    thereof the nucleic acid of any one of Items 1 to 73, the    composition of any one of Items 74 to 162, the polypeptide of Item    163, the vaccine of Item 164 to 166, and/or the kit or kit of parts    of Item 167.-   Item 171. The method of treating or preventing a disorder of Item    170, wherein the disorder is an infection with a coronavirus,    preferably a SARS-CoV-2 coronavirus, or a disorder related to such    an infection, preferably COVID-19.-   Item 172. The method of treating or preventing a disorder of Item    170 or 171, wherein the subject in need is a mammalian subject,    preferably a human subject.-   Item 173. The method of treating or preventing a disorder of any one    of Items 170 to 172, wherein the human subject is an elderly human    subject, preferably of an age of at least 50, 60, 65, or 70 years.-   Item 174. The method of treating or preventing a disorder of Item    173, wherein the human subject is 61 years of age or older.-   Item 175. The method of treating or preventing a disorder of any one    of Items 170 to 172, wherein the human subject is 18 to 60 years of    age.-   Item 176. The method of treating or preventing a disorder of any one    of Items 170 to 172, wherein the subject is pregnant.-   Item 177. The method of any one of Items 170 to 175, wherein no more    than 25% of subjects experience a Grade 3 systemic adverse event    after a first dose of the composition or wherein no more than 30% of    subjects experience a Grade 2 or higher local adverse event after a    first dose of the composition.-   Item 178. The method of any one of Items 170 to 175, wherein no more    than 40% of subjects experience a Grade 3 systemic adverse event    after a second dose of the composition.-   Item 179. The method of treating or preventing a disorder of any one    of Items 170 to 172, wherein the human subject is a newborn or    infant, preferably of an age of not more than 3 years, of not more    than 2 years, of not more than 1.5 years, of not more than 1 year    (12 months), of not more than 9 months, 6 months or 3 months, or of    an age between 6 months and 2 years-   Item 180. The method of Item 170, further defined as a method of    reducing disease burden in the subject.-   Item 181. The method of Item 180, wherein the method reduces the    severity of one or more symptom of COVID-19 disease.-   Item 182. The method of Item 181, wherein the method reduces the    probability that the subject will require hospital admission,    intensive care unit admission, treatment with supplemental oxygen    and/or treatment with a ventilator.-   Item 183. The method of Item 181, wherein the method reduces the    probability that the subject will develop severe or moderate    COVID-19 disease.-   Item 184. The method of Item 181, wherein the method prevents severe    COVID-19 disease in the subject for at least about 6 months.-   Item 185. The method of Item 184, wherein the method prevents severe    COVID-19 disease in the subject when the subject is exposed to a    SARS CoV-2 variant having a least a first amino acid change in the S    protein as compared to SEQ ID NO: 1.-   Item 186. The method of Item 185, wherein the SARS CoV-2 variant has    amino acid changes in the S protein comprising:    -   (i) delH69, delV70, Y453F, D614G, 1692V and M1229I;    -   (ii) delH69, delV70, delY144, N501Y, A570D, D614G, P681H, T716I,        S982A and D1118H;    -   (iii) L18F, D80A, D215G, delL242, delA243, delL244, R2461,        K417N, E484K, N501Y, D614G and A701V;    -   (iv) L18F, T20N, P26S, D138Y, R190S, K417T, E484K, N501Y, D614G,        H655Y and T10271; and/or    -   (v) S13I, W152C, L452R, and D614G.-   Item 187. The method of Item 181, wherein the method reduces the    probability that the subject will develop a fever, breathing    difficulties; loss of smell and/or loss of taste.-   Item 188. The method of Item 181, wherein the method reduces the    probability that the subject will develop a fever, breathing    difficulties; loss of smell and/or loss of taste.-   Item 189. The method of Item 170, wherein the subject has a disease    or is immune compromised.-   Item 190. The method of Item 189, wherein the subject has liver    disease, kidney disease diabetes, hypertension, heart disease, lung    disease, cancer or is HIV positive.-   Item 191. The method of Item 170, wherein the subject has not been    treated with an immunosuppressant drug for more than 14 days in the    last 6 months.-   Item 192. The method of Item 170, wherein the subject has not    received a live vaccine for at least 28 days prior to the    administration and/or has not received an inactivated vaccine for at    least 14 days prior to the administration.-   Item 193. A method of stimulating an immune response in a subject,    wherein the method comprises administering to the subject at least a    first composition comprising the nucleic acid, preferably mRNA of    any one of Items 1 to 73, the composition of any one of Items 74 to    162, the polypeptide of Item 163, the vaccine of Item 164 to 166,    and/or the kit or kit of parts of Item 167.-   Item 194. The method of Item 193, wherein the subject was previously    infected with SARS CoV-2.-   Item 195. The method of Item 193, wherein the subject was previously    treated with at least a first SARS CoV-2 vaccine composition.-   Item 196. The method of Item 195, wherein the first SARS CoV-2    vaccine composition was a mRNA vaccine.-   Item 197. The method of Item 196, wherein the first SARS CoV-2    vaccine composition was BNT162 or mRNA-1273.-   Item 198. The method of Item 195, wherein the first SARS CoV-2    vaccine composition was a protein subunit vaccine.-   Item 199. The method of Item 198, wherein the first SARS CoV-2    vaccine composition was NVX-CoV2373 or COVAX.-   Item 200. The method of Item 195, wherein the first SARS CoV-2    vaccine composition was an adenovirus vector vaccine.-   Item 201. The method of Item 200, wherein the first SARS CoV-2    vaccine composition was ADZ1222 or Ad26.COV-2.S.-   Item 202. The method of any one of Items 193-201, wherein the    subject has detectable SARS CoV-2 binding antibodies.-   Item 203. The method of Item 202, wherein the subject has detectable    SARS CoV-2 S protein-binding antibodies.-   Item 204. The method of Item 202, wherein the subject has detectable    SARS CoV-2 N protein-binding antibodies.-   Item 205. The method of any one of Items 195-201, wherein the first    SARS CoV-2 vaccine composition was administered to the patient at    least about 3 month, 6 months, 9 months, 1 year, 1.5 years, 2 years    or 3 years ago.-   Item 206. The method of any one of Items 195-201, wherein the first    SARS CoV-2 vaccine composition was administered to the patient    between about 3 months and 2 years ago or between about 6 months and    2 years ago.-   Item 207. The method of any one of Items 193-206, wherein the method    prevents moderate and severe COVID-19 disease in at least 80%, 85%,    90% or 95% of treated subjects.-   Item 208. The method of Item 207, wherein the method prevents    moderate and severe COVID-19 disease in at least 80%, 85%, 90% or    95% of treated subjects from about 2 weeks to about 1 year after    said administration.-   Item 209. The method of Item 207, wherein the method prevents    moderate and severe COVID-19 disease in at least 80%, 85%, 90% or    95% of treated subjects from about 2 weeks to about 3 month, 6    months, 9 months, 1 year, 1.5 years, 2 years or 3 years after said    administration.-   Item 210. The method of any one of Items 193-209, wherein the method    prevents SARS CoV-2 infection of the subject and/or SARS CoV-2    transmission from the subject in at least 50%, 55%, 60%, 65%, 70%,    75%, 80%, 85%, 90% or 95% of treated subjects.-   Item 211. The method of Item 210, wherein the prevents SARS CoV-2    infection of the subject and/or SARS CoV-2 transmission from the    subject in at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or    95% of treated subjects from about 2 weeks to about 1 year after    said administration.-   Item 212. The method of Item 211, wherein the method prevents SARS    CoV-2 infection of the subject and/or SARS CoV-2 transmission from    the subject in at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%    or 95% of treated subjects from about 2 weeks to about 3 month, 6    months, 9 months, 1 year, 1.5 years, 2 years or 3 years after said    administration.-   Item 213. The method of any one of Items 193-212, further comprising    administering at least a second composition to the subject, the    second composition comprising the nucleic acid, preferably mRNA of    any one of Items 1 to 61, the composition of any one of Items 74 to    128, the polypeptide of Item 163, the vaccine of Item 164 to 166,    and/or the kit or kit of parts of Item 167.-   Item 214. The method of Item 213, wherein the second composition is    administered at least about 7 days after said first composition.-   Item 215. The method of Item 214, wherein the second composition is    administered at least about 10 days, 14 days, 21 days, 28 days, 35    days, 42 days, 49 days or 56 days after said first composition.-   Item 216. The method of Item 213, wherein the second composition is    administered between about 7 days and about 56 days after said first    composition.-   Item 217. The method of Item 216, wherein the second composition is    administered between: about 14 days and about 56 days; about 21 days    and about 56 days; or about 28 days and about 56 days after said    first composition.-   Item 218. The method of any one of Items 193-212, further comprising    administering at least a third composition to the subject, the third    composition comprising the nucleic acid of any one of Items 1 to 61,    the composition of any one of Items 74 to 162, the polypeptide of    Item 163, the vaccine of Item 164 to 166, and/or the kit or kit of    parts of Item 167.-   Item 219. The method of any one of Items 213-218, wherein the method    prevents moderate and severe COVID-19 disease in at least 80%, 85%,    90% or 95% of treated subjects.-   Item 220. The method of Item 219, wherein the method prevents    moderate and severe COVID-19 disease in at least 80%, 85%, 90% or    95% of treated subjects from about 2 weeks to about 1 year after    administering the second or subsequent composition.-   Item 221. The method of Item 219, wherein the method prevents    moderate and severe COVID-19 disease in at least 80%, 85%, 90% or    95% of treated subjects from about 2 weeks to about 3 month, 6    months, 9 months, 1 year, 1.5 years, 2 years or 3 years after    administering the second or subsequent composition.-   Item 222. The method of any one of Items 193-221, further defined as    a method of stimulating an antibody, a CD4+ T cell response or a    CD8+ T-cell response in the subject.-   Item 223. The method of any one of Items 193-221, further defined as    a method of stimulating a neutralizing antibody response in the    subject.-   Item 224. The method of any one of Items 193-221, wherein the method    stimulates an antibody response that produces between about 10 and    about 500 coronavirus spike protein-binding antibodies for every    coronavirus neutralizing antibody in the subject.-   Item 225. The method of Item 224, wherein the method stimulates an    antibody response that produces no more than about 200 spike    protein-binding antibodies for every coronavirus neutralizing    antibody.-   Item 226. The method of Item 224, wherein the method stimulates an    antibody response that produces between about 10 and about 300;    about 20 and about 300; about 20 and about 200; or about 30 and    about 100 coronavirus spike protein-binding antibodies for every    coronavirus neutralizing antibody.-   Item 227. The method of Item 226, wherein the method stimulates an    antibody response that produces between about 30 and about 80    coronavirus spike protein-binding antibodies for every coronavirus    neutralizing antibody.-   Item 228. The method of Item 223, wherein the method stimulates an    antibody response that produces between about 1 and about 500    coronavirus spike protein receptor binding domain (RBD)-binding    antibodies for every coronavirus neutralizing antibody in the    subject.-   Item 229. The method of Item 228, wherein the method stimulates an    antibody response that produces no more than about 50 spike protein    RBD-binding antibodies for every coronavirus neutralizing antibody.-   Item 230. The method of Item 228, wherein the method stimulates an    antibody response that produces between about 1 and about 200; about    2 and about 100; about 3 and about 200; about 5 and about 100; or    about 5 and about 50 spike protein RBD-binding antibodies for every    coronavirus neutralizing antibody.-   Item 231. The method of Item 230, wherein the method stimulates an    antibody response that produces between about 5 and about 20    coronavirus spike protein RBD-binding antibodies for every    coronavirus neutralizing antibody.-   Item 232. The method of Item 222, wherein the subject has been    previously infected with SARS-CoV-2.-   Item 233. The method of Item 222, further defined as a method    stimulating a protective immune response in the subject.-   Item 234. The method of any one of Items 193-233, wherein the    subject is a human subject.-   Item 235. The method of Item 234, wherein the subject is between the    ages of 6 months and 100 years, 6 months and 80 years, 1 year and 80    years, 1 year and 70 years, 2 years and 80 years or 2 years and 60    years.-   Item 236. The method of Item 234, wherein the subject is a newborn    or infant of an age of not more than 3 years, of not more than 2    years, of not more than 1.5 years, of not more than 1 year (12    months), of not more than 9 months, 6 months or 3 months, or is    between 6 months and 2 years.-   Item 237. The method of Item 234, wherein the subject is an elderly    subject of an age of at least 50, 60, 65, or 70 years.-   Item 238. The method of Item 237, wherein the subject is an elderly    subject of an age of at least 60 years.-   Item 239. The method of any one of Items 234 to 238, wherein the    subject has native American, African, Asian or European heritage.-   Item 240. The method of Item 238, wherein the subject has at least    about 10%, 25%, or 50% native American, African, Asian or European    heritage.-   Item 241. The method of Item 238, wherein the subject has native    American heritage.-   Item 242. The method of Item 238, wherein the subject has at least    about 10%, 25% or 50% native American heritage.-   Item 243. The method of any one of Items 193-242, wherein the method    induces essentially no increase in Th2 cytokines, preferably IL-4,    IL-13, TNF and/or IL-1β in the subject-   Item 244. The method of any one of Items 193-242, further defined as    a method of inducing a Th1 directed immune response in the subject.-   Item 245. The method of any one of Items 193-244, wherein the    subject is receiving anti-coagulation therapy.-   Item 246. The method of any one of Items 193-245, wherein the    composition is administered by intramuscular injection.-   Item 247. The method of any one of Items 193-246, wherein the    composition comprises a mRNA encoding a coronavirus spike    protein (S) that is a pre-fusion stabilized spike protein (S_stab)    comprising at least one pre-fusion stabilizing mutation.-   Item 248. The method of Item 247, wherein the mRNA encodes a    coronavirus spike protein at least 95% identical to SEQ ID NO: 163.-   Item 249. The method of Item 248, wherein the mRNA encodes a    coronavirus spike protein identical to SEQ ID NO: 163.-   Item 250. The method of Item 247, wherein the mRNA encodes a    coronavirus spike protein at least 95% identical to SEQ ID NO: 149.-   Item 251. The method of Item 248, wherein the mRNA encodes a    coronavirus spike protein identical to SEQ ID NO: 149.-   Item 252. The method of Item 250 or 251, wherein a single dose of    the composition provides a sufficient immune response to protect the    subject from severe COVID-19 disease for at least about 6 months.-   Item 253. The method of Item 252, wherein a single dose of the    composition provides a sufficient immune response to protect the    subject from severe COVID-19 disease for about 6 months to about 1    year, 1.5 years, 2 years, 2.5 years, 3 years, 4 years or 5 years.-   Item 254. The method of any one of Items 247-249, wherein the mRNA    is complexed with one or more lipids thereby forming LNP.-   Item 255. The method of Item 254, wherein the LNP comprises    -   (i) at least one cationic lipid;    -   (ii) at least one neutral lipid;    -   (iii) at least one steroid or steroid analogue; and    -   (iv) at least one PEG-lipid,    -   wherein (i) to (iv) are in a molar ratio of about 20-60%        cationic lipid, 5-25% neutral lipid, 25-55% sterol, and 0.5-15%        PEG-lipid.-   Item 256. The method of Item 255, wherein the LNP comprises    -   (i) at least one cationic lipid according to formula III-3;    -   (ii) DSPC;    -   (iii) cholesterol; and    -   (iv) a PEG-lipid, according to formula IVa,    -   wherein (i) to (iv) are in a molar ratio of about 20-60%        cationic lipid, 5-25% neutral lipid, 25-55% sterol, and 0.5-15%        PEG-lipid.-   Item 257. The method of Item 256, wherein the LNP comprises    -   (i) at least one cationic lipid according to formula III-3;    -   (ii) DSPC;    -   (iii) cholesterol; and    -   (iv) a PEG-lipid, according to formula IVa,    -   wherein (i) to (iv) are in a molar ratio of about        47.5:10:40.8:1.7.-   Item 258. The method of Item 256, wherein the LNP comprises    -   (i) at least one cationic lipid according to formula III-3;    -   (ii) DSPC;    -   (iii) cholesterol; and    -   (iv) a PEG-lipid, according to formula IVa,    -   wherein (i) to (iv) are in a molar ratio of 47.4:10:40.9:1.7.-   Item 259. The method of anyone of Items 254-258, wherein the ratio    of mRNA to total lipid is about 0.03-0.04 w/w.-   Item 260. The method of Item 249, wherein the mRNA is complexed with    one or more lipids thereby forming lipid nanoparticles (LNP),    wherein the LNP comprises    -   (i) at least one cationic lipid according to formula III-3;    -   (ii) DSPC;    -   (iii) cholesterol; and    -   (iv) a PEG-lipid, according to formula IVa,    -   wherein (i) to (iv) are in a molar ratio of about        47.5:10:40.8:1.7, and    -   wherein the ratio of mRNA to total lipid is about 0.03-0.04 w/w.-   Item 261. The method of Item 249, wherein the mRNA is complexed with    one or more lipids thereby forming lipid nanoparticles (LNP),    wherein the LNP comprises    -   (i) at least one cationic lipid according to formula III-3;    -   (ii) DSPC;    -   (iii) cholesterol; and    -   (iv) a PEG-lipid, according to formula IVa,    -   wherein (i) to (iv) are in a molar ratio of 47.4:10:40.9:1.7,        and    -   wherein the ratio of mRNA to total lipid is about 0.03-0.04 w/w.-   Item 262. The method of any one of Items 247-261, wherein the    subject is administered a composition that comprises between about 2    μg and about 50 μg of mRNA.-   Item 263. The method of Item 262, wherein the subject is    administered a composition that between about 10 μg and about 50 μg    of mRNA.-   Item 264. The method of Item 263, wherein the subject is    administered a composition that between about 10 μg and about 30 μg    of mRNA.-   Item 265. The method of Item 264, wherein the subject is    administered a composition that comprises about 12 μg of mRNA.-   Item 266. The method of any one of Items 264 to 265, wherein the    administration provides seroconversion in 100% of subjects to which    the composition is administered.-   Item 267. The method of any one of Items 193 to 266, wherein the    human subject is 61 years of age or older.-   Item 268. The method of any one of Items 193 to 266, wherein the    human subject is 18 to 60 years of age.-   Item 269. The method of any one of Items 193 to 268, wherein the    human subject has had a previous vaccine allergy.-   Item 270. The method of any one of Items 193 to 269, wherein the    subject has detectable anti-PEG antibodies.-   Item 271. The method of the any one of Items 193 to 270 comprising:    -   (i) obtaining a composition of any one of Items 74 to 162,        wherein the composition is lyophilized;    -   (ii) solubilizing the lyophilized composition in a        pharmaceutically acceptable liquid carrier to produce a liquid        composition; and    -   (iii) administering an effective amount of the liquid        composition to the subject.-   Item 272. A method of stabilizing a composition of any one of Items    74 to 162 comprising lyophilizing the composition to a produce a    stabilized composition.-   Item 273. The method of Item 272, wherein the stabilized composition    has a water content of less than about 10%.-   Item 274. The method of Item 273, wherein the stabilized composition    has a water content of between about 0.5% and 5.0%.-   Item 275. A stabilized, lyophilized composition produced by a method    of any one of Items 272-274.

Brief Description of Lists and Tables

-   List A: Exemplary SARS-CoV-2 coronavirus isolates-   List B: GenBank Accession Numbers of different SARS-CoV-2 isolates-   List 1: Exemplary suitable protein designs of the invention-   Table A: Intensity Grading for Solicited Local Adverse Events-   Table B: Intensity Grading for Solicited Systemic Adverse Events-   Table 1: Preferred coronavirus constructs (amino acid sequences and    nucleic acid coding sequences)-   Table 2: Human codon usage table with frequencies indicated for each    amino acid-   Table 3a: Nucleic acid, preferably mRNA constructs suitable for a    coronavirus vaccine-   Table 3b: Nucleic acid, preferably mRNA constructs suitable for a    coronavirus vaccine-   Table 4: RNA constructs encoding different SARS-CoV-2 S antigen    design (used in the Examples)-   Table A: Lipid-based carrier composition of the examples-   Table 5: Overview of mRNA constructs used in Example 2a-   Table 6: Overview of mRNA constructs used in Example 2b-   Table 7: Overview of mRNA constructs used in Example 2c-   Table 8: Vaccination regimen (Example 3)-   Table 9: Vaccination regimen (Example 4)-   Table 10: Vaccination regimen (Example 5)-   Table 11: Vaccination regimen (Example 6)-   Table 12: Vaccination regimen (Example 7)-   Table 13: Vaccination regimen (Example 8)-   Table 14: Vaccination regimen (Example 9)-   Table 15: List of histopathological analysis indicated in FIG. 12F:-   Table 16: Vaccination regimen (Example 11)-   Table 17: Vaccination regimen (Example 12)-   Table 18: Primary and Supportive Populations for the Analysis of    Each Endpoint-   Table 19: Two Stage Group Sequential Design with Interim Analyses at    56 and 111 Cases and Final Analysis at 185 Cases-   Table 20: Vaccination regimen (Example 14)-   Table 21: Vaccination regimen (Example 15)-   Table 22: Vaccination regimen (Example 16)-   Table 23: Vaccination regimen (Example 17)-   Table 24: Vaccination regimen (Example 18)-   Table 25: List of emerging SARS-CoV-2 isolates/variants (Example 19)

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows that mRNA constructs encoding different SARS-CoV-2 Sprotein designs led to a detectable protein expression using an in vitrotranslation system. Further details provided in Example 2a and Table 5.

FIG. 2 shows that mRNA constructs encoding different SARS-CoV-2 Sprotein designs are expressed on the cell surface of mammalian cellsusing FACS analysis. Further details provided in Example 2b and Table 6.

FIG. 3 shows that mRNA constructs encoding different SARS-CoV-2 Sproteins are expressed in mammalian cells using western blot analysis.Further details provided in Example 2c and Table 7.

FIG. 4 shows significant IgG1 and IgG2a responses for group vaccinatedwith the mRNA vaccine encoding full length stabilized S protein. FIG. 4shows comparable IgG1 response for the mRNA vaccine and the rec.SARS-CoV-S protein and higher IgG2a titers for the mRNA vaccine comparedto the rec. SARS-CoV-S protein. IgG1 and IgG2a antibody titers assessedby ELISA using rec. SARS-CoV-2 S protein as a coating reagent. Theexperiment was performed as described in Example 5. Further constructdetails are provided in Table 10.

FIGS. 5A-C show significant IgG1 and IgG2a responses for groupvaccinated with the mRNA vaccine encoding full length stabilized Sprotein and full length wildtype S protein. FIG. 5A shows comparableIgG1 response for both full length S protein designs and FIG. 5B showscomparable IgG2a titers for both full length S protein designs. FIG. 5Cshows high and comparable virus neutralizing titers for both full-lengthS protein designs at day 42. The experiments were performed as describedin Example 6. Further construct details are provided in Table 11.

FIG. 6 shows that LNP formulated mRNA encoding full length stabilized Sprotein and full length S protein induces cellular immune responses inmice (CD8+ and/or CD4+ T cell responses), using an intracellularcytokine staining assay. Groups A-C LNP formulated mRNA encodingdifferent full-length S protein designs; Group D negative control.Vaccination scheme see Table 11. Further details provided in Example 6.

FIG. 7 shows innate immune responses after vaccination with LNPformulated mRNA encoding full-length S protein (S_stab) (group A). Thedotted lines indicate the lower limit of detection. The experiment wasperformed as described in Example 7. Further construct details areprovided in Table 12.

FIG. 8A shows significant IgG1 and IgG2a responses for groups vaccinatedwith the mRNA vaccine encoding full length stabilized S protein. FIG. 8Ashows high IgG1 responses and high IgG2a responses after firstvaccination. Groups A-D: one vaccination with LNP formulated mRNAencoding full-length S protein (S_stab); Group I: adjuvanted recombinantspike protein and Group J: negative control. The experiments wereperformed as described in Example 7. Further construct details areprovided in Table 12.

FIG. 8B shows significant IgG1 and IgG2a responses for all groupsvaccinated with the mRNA vaccine encoding full length stabilized Sprotein (s_stab). FIG. 8B (A) shows IgG1 response at day 28 (after firstvaccination) and at day 35 (after second vaccination) and FIG. 8B (B)shows IgG2a response at day 28 (after first vaccination) and at day 35(after second vaccination). Groups A-H: LNP formulated full-length Sprotein mRNA with different vaccination intervals; Group I: adjuvantedrecombinant spike protein and Group J: negative control. The experimentswere performed as described in Example 7. Further construct details areprovided in Table 12.

FIGS. 9A-B show significant induction of virus neutralizing titers (VNT)for all groups vaccinated with the mRNA vaccine encoding full lengthstabilized S protein (S_stab). FIG. 9A shows VNT at day 28 (after firstvaccination) and at day 35 and at day 49 (FIG. 9B) (after secondvaccination) Groups A-H: LNP formulated full length S protein mRNA withdifferent vaccination intervals; Group I: adjuvanted recombinant spikeprotein and Group J: negative control. The experiments were performed asdescribed in Example 7. Further construct details are provided in Table12.

FIG. 10 shows that LNP formulated mRNA encoding full length stabilized Sprotein (S_stab) induces cellular immune responses in mice (CD8+ and/orCD4+ T-cell responses) after second vaccination with different timeintervals between prime and boost vaccination, using an intracellularcytokine staining assay. Groups A-H: LNP formulated full length Sprotein mRNA with different vaccination intervals; Group I: adjuvantedrecombinant spike protein and Group J: negative control. The experimentswere performed as described in Example 7. Further construct details areprovided in Table 12.

FIGS. 11A-G show significant antibody responses in rats for groupsvaccinated with different doses of the mRNA vaccine encoding full lengthstabilized S protein (S_stab). FIG. 11A shows high IgG1 responses forgroups C-F, FIG. 11B shows high IgG2a responses for groups D-F and FIG.11C shows high total IgG response for groups C-E. Groups B-F: differentdoses of LNP formulated full length S protein mRNA and group A: negativecontrol. FIG. 11D shows further increased IgG1 antibody responses andFIG. 11E shows further increased IgG2a antibody titers for all groupsafter second vaccination at day 42. FIGS. 11F and G show thatvaccination with mRNA full length S stabilized protein formulated inLNPs induced in rats dose dependent levels of VNTs. The experiments wereperformed as described in Example 8. Further construct details areprovided in Table 13.

FIGS. 12A-F show protection of hamsters from SARS-CoV-2 challengevaccinated with different the inventive mRNA vaccine encoding fulllength stabilized S protein (S_stab). FIG. 12A shows the induction ofhigh total IgG antibodies for vaccinated groups E and F and FIG. 12Bshows the dose-dependent induction of VNTs upon one (day 28) or twovaccinations (day 42 and day 56). FIGS. 12C-E show detectable levels ofreplication competent virus in throat swabs on days 56 to day 60 (FIG.12C), nasal turbinate on day 60 (FIG. 12D) and lung tissues on day 60(FIG. 12F). Each dot represents an individual animal, bars depict themedian. Statistical analysis was performed using Mann-Whitney testing.FIG. 12F shows the protection of the respiratory tract of vaccinatedhamsters from challenge infection in the absence of signs of vaccineenhanced disease. Histopathological analysis was performed on day 60,four days post challenge infection, on formalin-fixed, paraffin embeddedtissues sections. Histopathological assessment scoring was performedaccording to severity of inspected parameter. Each dot represents anindividual animal, bars depict the median, Statistical analysis wasperformed using Mann-Whitney testing. The experiments were performed asdescribed in Example 9. Further details are provided in Table 14 andTable 15.

FIGS. 13A-K show the results of a phase I clinical trial in healthyhuman subjects. In FIG. 13A systemic adverse events are shown in thedifferent dose cohorts after the first dose and after the second dose.In FIG. 13B local adverse events are shown in the different dose cohortsafter the first dose and after the second dose. In FIG. 13C the specificsystemic adverse events are shown, such as fatigue, headache, myalgia,chills, arthralgia, fever, nausea and diarrhea. In FIG. 13D the specificlocal adverse events are shown, such as pain, itching, swelling andredness. In FIG. 13E induction of Spike protein specific IgG antibodieson day 1, 29, 36, 43 and 57 is shown for the different dose cohorts. Inthe table of FIG. 13E percentage of seroconversion of the vaccinatedsubjects is shown. In FIG. 13F induction of RBD-specific IgG antibodieson day 1, 36, and 43 is shown for the different dose cohorts. In thetable of FIG. 13F percentage of seroconversion of the vaccinatedsubjects is shown. In FIG. 13G induction of virus neutralizingantibodies is shown. In the table of FIG. 13G percentage ofseroconversion of the vaccinated subjects is shown. In FIG. 13H theratios of the level of Spike protein or RBD binding antibodies to thelevel of neutralizing antibodies are shown. FIG. 13I shows induction ofCD4+ T cells against Spike protein S1 after the first dose (day 29) andthe second dose (day 36). FIG. 13J shows induction of CD4+ T cellsagainst Spike protein S2 after the first dose (day 29) and the seconddose (day 36). In FIG. 13K induction of virus neutralizing titers andRBD specific antibodies in SARS-CoV-2 seropositive subjects aftervaccination with 2 μg and 4 μg CvnCoV is shown.

FIGS. 14A-C show significant IgG1 and IgG2a responses after thevaccination with mRNA encoding full length S stabilized protein (S_stab)after a single vaccination (d21) and more increased after a secondvaccination (d42) (FIG. 14A). Vaccine composition comprising mRNAencoding SARS-CoV-2 S_stab comprising hSL-A100 and the UTR combinationa-1 (HSD17B4/PSMB3) (group C) shows an improved and stronger inductionof binding antibodies (shown by IgG1 and IgG2a endpoint titers). Theinduction of VNT is shown in FIG. 14B. Mice of group C showed an earlyincreased level of VNTs already on d21 after first vaccination comparedto group B. The induction of T-cell immunity is shown in FIG. 14C.Vaccine composition comprising mRNA encoding SARS-CoV-2 S_stabcomprising hSL-A100 and the UTR combination a-1 (HSD17B4/PSMB3) (groupC) shows surprisingly a remarkable stronger induction of CD8⁺ IFNγ/TNFdouble positive T cells.

FIGS. 15A-B show significant antibody responses in rats for groupsvaccinated with different doses of the mRNA vaccine encoding full lengthstabilized S protein (S_stab) comprising the alternative non-codingregion with 3′ end hSL-A100 and the UTR combination a-1 (HSD17B4/PSMB3)formulated in LNPs. FIG. 15A shows a robust induction of IgG1 and IgG2abinding antibodies and FIG. 15B the induction of VNTs in adose-dependent manner. The experiments were performed as described inExample 12. Further construct details are provided in Table 17.

FIGS. 16A-C show significant antibody responses in rats for groupsvaccinated with different doses of the mRNA vaccine encoding full lengthstabilized S protein (S_stab) comprising different non-coding regionsformulated in LNPs. FIG. 16A shows a robust and dose-dependent inductionof IgG1 and IgG2a binding antibodies and FIG. 16B the early induction ofVNTs after only one dose of vaccination in a dose-dependent manner forthe mRNA vaccine encoding full length stabilized S protein (S_stab)comprising the non-coding region with 3′ end hSL-A100 and the UTRcombination a-1 (HSD17B4/PSMB3) formulated in LNPs. FIG. 16C shows theinduction of VNTs after two doses of vaccination on day 42. Theexperiments were performed as described in Example 14. Further constructdetails are provided in Table 20.

FIGS. 17A-D show that CVnCoV (mRNA vaccine encoding full lengthstabilized S protein (S_stab) formulated in LNPs) induces humoralresponse in non-human primates. (FIG. 17A) Schematic drawing of studysetup. Rhesus macaques (n=6; 3 male, 3 female/group) were vaccinated IMon day 0 and day 28 with 0.5 μg or 8 μg of CVnCoV or remainedunvaccinated. All animals were challenge with 5.0×10⁶ PFU of SARS-CoV-2on d56. Two animals of each group were terminated on d62, d63 and d64,respectively. (FIG. 17B) Trimeric Spike protein or (FIG. 17C) RBDspecific binding IgG antibodies, displayed as endpoint titres atdifferent time points as indicated. (FIG. 17D) Virus neutralisingantibodies determined via focus reduction neutralisation test atdifferent time points as indicated. All values are displayed as medianwith range. Dotted lines represent vaccinations and challenge infection,respectively. RBD receptor binding domain; VNT virus neutralising titre.The experiment was performed as described in Example 15. Furtherconstruct details are provided in Table 21.

FIGS. 18A-B show that CVnCoV (mRNA vaccine encoding full lengthstabilized S protein (S_stab) formulated in LNPs) induces cellularresponses in non-human primates. PBMCs from 0.5 μg or 8 μg CVnCoVvaccinated or from untreated animals isolated at different time pointswere re-stimulated with S specific peptide pools ex vivo followed byIFNγELISpot analysis. (FIG. 18A) IFNγELISpot before challenge infectionon d56. Panel 1 represent results of stimulation with a single peptidepool covering the whole S protein, panels 2-4 depict stimulation resultsof ten individual pools covering the entire S protein in each group.(FIG. 18B) IFNγELISpot until termination on d62-d64. Panel 1 representresults of stimulation with three megapools and shows the mummedresponse covering the whole S protein, panels 2-4 depict stimulationresults of ten individual pools covering the entire S protein in eachgroup. SFU spot forming unit; PP peptide pool. The experiment wasperformed as described in Example 15. Further construct details areprovided in Table 21.

FIGS. 19A-F show that CVnCoV (mRNA vaccine encoding full lengthstabilized S protein (S_stab) formulated in LNPs) protects non-humanprimates from challenge infection (FIG. 19A) Nasal swabs taken atdifferent time points post challenge (FIG. 19B) in life BAL samplestaken on d59 and at termination on d62-64 and (FIG. 19C) lung tissuehomogenates from d62-64 were analysed for copies of total viral RNA viaRT-qPCR. (FIG. 19D) Nasal swabs taken at different time points pastchallenge (FIG. 19E) in life BAL samples taken on d59 and at terminationon d62-64 and (FIG. 19F) lung tissue homogenates from d62-64 wereanalysed for copies of subgenomic viral RNA via RT-qPCR. Values aredepicted as medians with range. Lower and upper dotted lines representLLOD and LLOQ, respectively. Kruskall-Wallis ANOVA followed by Dunn'stest was used to compare groups and P values are shown. LLOD lower limitof detection, LLOQ lower limit of quantification, RT-qPCR Reversetranscription-quantitative polymerase chain reaction. The experiment wasperformed as described in Example 15. Further construct details areprovided in Table 21.

FIGS. 20A-1 show that CVnCoV (mRNA vaccine encoding full lengthstabilized S protein (S_stab) formulated in LNPs) protects non-humanprimates from challenge infection (FIG. 20A) Throat swabs taken atdifferent time points past challenge were analysed for copies of totalviral RNA via RT-qPCR (FIG. 20B) Throat swabs taken at different timepoints past challenge analysed for copies of subgenomic RNA via RT-qPCR.Homogenised tissue derived from (FIG. 20C) tonsils (FIG. 20D) trachea(FIG. 20E) spleen (FIG. 20F) duodenum (FIG. 20G) colon (FIG. 20H) liver(FIG. 20I) kidney were analysed for copies of total viral RNA viaRT-qPCR. (RT-qPCR Reverse transcription-quantitative polymerase chainreaction, sg subgenomic). The experiment was performed as described inExample 15. Further construct details are provided in Table 21.

FIGS. 21A-F Exemplary sections showing histopathology (H&E) andSARS-CoV-2 in situ hybridisation (ISH). FIG. 21A: Alveolar necrosis andinflammatory exudates (*) in the alveolar spaces and type II pneumocytehyperplasia (arrows). FIG. 21B: Mild perivascular cuffing (arrow). FIG.21C: Inflammatory cell infiltration in the alveolar spaces and theinteralveolar septa (*) and type II pneumocyte hyperplasia (arrows).FIG. 21D: SARS-CoV-2 ISH staining in abundant cell within inflammatoryfoci (arrows). FIG. 21E: SARS-CoV-2 ISH staining in a single cell withinan interalveolar septum (arrow). FIG. 21F: Abundant foci of SARS-CoV-2ISH stained cells within the alveolar lining and the interalveolar septa(arrows) (Bar=100 μm. ISH in situ hybridisations). The experiment wasperformed as described in Example 15. Further construct details areprovided in Table 21.

FIGS. 22A-D show that vaccination with 8 μg of CVnCoV (mRNA vaccineencoding full length stabilized S protein (S_stab) formulated in LNPs)protects the lungs from pathological changes upon viral challenge (FIG.22A) Heat map showing scores for each lung pathology parameter and theaverage score for each animal from all groups as indicated. Severityranges from 0 to 4: 0=none; 1=minimal; 2=mild; 3=moderate and4=marked/severe. (FIG. 22B) Graph representing the cumulative score forall the lung histopathology parameters from each animal. (FIG. 22C)Presence of viral RNA in lung tissue sections from all animals expressedas percentage of ISH (RNAScope, in situ hybridisation) positive stainingarea of lung section. (FIG. 22D) Cumulative score of lung pathologydetected via CT radiology. Box and whiskers indicate median with range.Kruskall-Wallis ANOVA followed by Dunn's test was used to compare groupsand P values are shown. The experiment was performed as described inExample 15. Further construct details are provided in Table 21.

FIGS. 23A-C show induction of IFNa in human PBMCs (FIG. 23A) stimulatedwith mRNA vaccine compositions. Induction of VNTs after one vaccinationonly (on day 21) and after two vaccination (on day 42) is shown in FIGS.23B and C. All of the mRNA vaccine compositions with mRNAs comprising a3′ end “hSL-A100” or “A-100” (groups C-G, I-M) showed improved, earlyand strong induction of VNTs. In these constructs, the poly(A) sequenceis located directly at the 3′ terminus of the RNA.

FIGS. 24A-B FIG. 24A shows the induction of VNTs after only onevaccination. mRNA vaccine compositions with mRNAs comprising a 3′ end“hSL-A100” or “A-100” showed improved, early and strong induction ofVNTs. In these constructs, the poly(A) sequence is located directly atthe 3′ terminus of the RNA. FIG. 24B demonstrate the induction of VNTsafter only one vaccination (group A-E) or after two vaccination (groupF-J) at a later timepoint on day 42. mRNA vaccine composition comprisingR9709 (group B) induced most prominent titers of VNTs between the groupsreceiving only one vaccination.

EXAMPLES

In the following, particular examples illustrating various embodimentsand aspects of the invention are presented. However, the presentinvention shall not to be limited in scope by the specific embodimentsdescribed herein. The following preparations and examples are given toenable those skilled in the art to more clearly understand and topractice the present invention. The present invention, however, is notlimited in scope by the exemplified embodiments, which are intended asillustrations of single aspects of the invention only, and methods,which are functionally equivalent are within the scope of the invention.Indeed, various modifications of the invention in addition to thosedescribed herein will become readily apparent to those skilled in theart from the foregoing description, accompanying figures and theexamples below. All such modifications fall within the scope of theappended claims.

Example 1: Preparation of DNA and RNA Constructs, Compositions, andVaccines

The present Example provides methods of obtaining the RNA of theinvention as well as methods of generating a composition or a vaccine ofthe invention.

1.1. Preparation of DNA and RNA Constructs:

DNA sequences encoding different SARS-CoV-2 S protein designs wereprepared and used for subsequent RNA in vitro transcription reactions.Said DNA sequences were prepared by modifying the wild type or referenceencoding DNA sequences by introducing a G/C optimized or modified codingsequence (e.g., “cds opt1”) for stabilization and expressionoptimization. Sequences were introduced into a pUC derived DNA vector tocomprise stabilizing 3′-UTR sequences and 5′-UTR sequences, additionallycomprising a stretch of adenosines (e.g. A64 or A100), and optionally ahistone-stem-loop (hSL) structure, and optionally a stretch of 30cytosines (e.g. C30) (see Table 4, for an overview of coronavirusantigen designs see List 1 or Table 1).

The obtained plasmid DNA constructs were transformed and propagated inbacteria using common protocols known in the art. Eventually, theplasmid DNA constructs were extracted, purified, and used for subsequentRNA in vitro transcription (see section 1.2).

Alternatively, DNA plasmids can be used as template forPCR-amplification (see section 1.3).

1.2. RNA In Vitro Transcription from Plasmid DNA Templates:

DNA plasmids prepared according to section 1.1 were enzymaticallylinearized using a restriction enzyme and used for DNA dependent RNA invitro transcription using T7 RNA polymerase in the presence of anucleotide mixture (ATP/GTP/CTP/UTP) and cap analog (e.g. m7GpppG,m7G(5′)ppp(5′)(2′OMeA)pG, m7G(5′)ppp(5′)(2′OMeG)pG), or3′OMe-m7G(5′)ppp(5′)(2′OMeA)pG) under suitable buffer conditions. Theobtained RNA constructs were purified using RP-HPLC (PureMessenger®,CureVac AG, Tubingen, Germany; WO2008/077592) and used for in vitro andin vivo experiments. DNA templates may also be generated using PCR. SuchPCR templates can be used for DNA dependent RNA in vitro transcriptionusing an RNA polymerase as outlined herein.

To obtain chemically modified mRNA, RNA in vitro transcription wasperformed in the presence of a modified nucleotide mixture comprisingpseudouridine N(1)-methylpseudouridine (m1ψ) instead of uracil. Theobtained m1ψ chemically modified RNA was purified using RP-HPLC(PureMessenger®, CureVac AG, Tubingen, Germany; WO2008/077592) and usedfor further experiments (see e.g. Example 16 or 17).

Generation of Capped RNA Using Enzymatic Capping (Prophetic):

Some RNA constructs are in vitro transcribed in the absence of a capanalog. The cap-structure (cap0 or cap1) is then added enzymaticallyusing capping enzymes as commonly known in the art. In short, in vitrotranscribed RNA is capped using a capping kit to obtain cap0-RNA.cap0-RNA is additionally modified using cap specific2′-O-methyltransferase to obtain cap1-RNA. cap1-RNA is purified e.g. asexplained above and used for further experiments.

RNA for clinical development is produced under current goodmanufacturing practice e.g. according to WO2016/180430, implementingvarious quality control steps on DNA and RNA level.

The RNA Constructs of the Examples:

The generated RNA sequences/constructs are provided in Table 4 with theencoded antigenic protein and the respective UTR elements indicatedtherein. If not indicated otherwise, the RNA sequences/constructs ofTable 4 have been produced using RNA in vitro transcription in thepresence of a m7GpppG, m7G(5′)ppp(5′)(2′OMeA)pG; accordingly, the RNAsequences/constructs comprise a 5′ Cap1 structure. If not indicatedotherwise, the RNA sequences/constructs of Table 4 have been produced inthe absence of chemically modified nucleotides (e.g. pseudouridine (ψ)or N(1)-methylpseudouridine (m1ψ)).

1.3. RNA In Vitro Transcription from PCR Amplified DNA Templates(Prophetic):

Purified PCR amplified DNA templates prepared according to paragraph 1.1is transcribed in vitro using DNA dependent T7 RNA polymerase in thepresence of a nucleotide mixture (ATP/GTP/CTP/UTP) and cap analog(m7GpppG or 3′-O-Me-m7G(5′)ppp(5′)G)) under suitable buffer conditions.Alternatively, PCR amplified DNA is transcribed in vitro using DNAdependent T7 RNA polymerase in the presence of a modified nucleotidemixture (ATP, GTP, CTP, N1-methylpseudouridine (m1ψ) or pseudouridine(ψ) and cap analogue (m7GpppG, m7G(5′)ppp(5′)(2′OMeA)pG orm7G(5′)ppp(5′)(2′OMeG)pG) under suitable buffer conditions. Some RNAconstructs are in vitro transcribed in the absence of a cap analog andthe cap-structure (cap0 or cap1) is added enzymatically using cappingenzymes as commonly known in the art. The obtained RNA is purified e.g.as explained above and used for further experiments. The obtained mRNAsare purified e.g. using RP-HPLC (PureMessenger®, CureVac AG, Tubingen,Germany; WO2008/077592) and used for in vitro and in vivo experiments.

TABLE 4 RNA constructs encoding different SARS-CoV-2 S antigen designs5′-UTR/ 3′-UTR; SEQ ID SEQ ID SEQ ID CDS UTR NO: NO: NO: RNA ID NameShort name opt. Design 3′-end Protein CDS RNA R9488, Spike S opt1H5D17B4/ hSL-A100 1 136 148 R9492, protein (gc) PSMB3; R10161* a-1 R9514Spike S opt1 —/muag; A64-N5- 1 136 162 protein (gc) i-3 C30-hSL- N5R9487, Spike pre- S_stab_PP opt1 HSD17B4/ hSL-A100 10 137 149 R9491,fusion (K986P_V987P) (gc) PSMB3; R9709, stabilized a-1 R10159**, proteinR10160* R9515, Spike pre- S_stab_PP opt1 —/muag; A64-N5- 10 137 163R10157** fusion (K986P_V987P) (gc) i-3 C30-hSL- stabilized N5 proteinR9486, Spike pre- S_stab_PP opt3 HSD17B4/ hSL-A100 10 142 150 R9490fusion (K986P_V987P) (human) PSMB3; stabilized a-1 protein R9517 Spikepre- S_stab_PP opt3 —/muag; A64-N5- 10 142 164 fusion (K986P_V987P)(human) i-3 C30-hSL- stabilized N5 protein R9519 Spike pre- S_stab_PPopt10 —/muag; A64-N5- 10 146 165 fusion (K986P_V987P) (gc i-3 C30-hSL-stabilized mod) N5 protein R9489, S fragment S1 opt1 HSD17B4/ hSL-A10027 138 152 R9493 (1-681) (gc) PSMB3; spike a-1 protein R9506, S fragmentS1 opt1 —/muag; A64-N5- 27 138 166 R9513 (1-681) (gc) i-3 C30-hSL- spikeN5 protein R9516 S fragment S1 opt3 —/muag; A64-N5- 27 143 167 (1-681)(human) 1-3 C30-hSL- spike N5 protein R9518 S fragment S1 opt10 —/muag;A64-N5- 27 147 168 (1-681) (gc i-3 C30-hSL- spike mod) N5 protein R9561Spike pre- S_stab_disul opt1 —/muag; A64-N5- 21 11804 12816 fusion(P7150_P1069C) (gc) i-3 C30-hSL- stabilized N5 protein R9564 Spike pre-S_stab_disul opt1 —/muag; A64-N5- 22 11805 12817 fusion (G8890_L1034C)(gc) i-3 C30-hSL- stabilized N5 protein R9562 Spike pre- S_stab_disulopt1 —/muag; A64-N5- 25 11808 12820 fusion (F9700_G999C) (gc) i-3C30-hSL- stabilized N5 protein R9560 Spike pre- S_stab_disul opt1—/muag; A64-N5- 1145 11810 12822 fusion (A8900_V1040C) (gc) i-3 C30-hSL-stabilized N5 protein R9563 Spike pre- S_stab_disul opt1 —/muag; A64-N5-1212 11811 12823 fusion (T8740_S1055C) (gc) i-3 C30-hSL- stabilized N5protein R9641 Spike pre- S_stab_PP_cav opt1 —/muag; A64-N5- 408 1179912811 fusion (K986P_V987P_ (gc) i-3 C30-hSL- stabilized T887W_A1020W) N5protein R9660 Spike pre- S_stab_PP_cav opt1 —/muag; A64-N5- 475 1180012812 fusion (K986P_V987P_ (gc) i-3 C30-hSL- stabilized P1069F) N5protein R9661 Spike pre- S_stab_PP_prot opt1 —/muag; A64-N5- 542 1180112813 fusion (K986P_V987P_ (gc) i-3 C30-hSL- stabilized H1048Q_H1064N_N5 protein H1083N_H1101N) R9663 Spike pre- S_stab_PP_ opt1 —/muag;A64-N5- 10726 11953 12931 fusion delTMflex_WhcAg (gc) i-3 C30-hSL-stabilized (K986P_V987P) N5 protein R9664 Spike pre- S_stab_PP_ opt1—/muag; A64-N5- 8716 11923 12901 fusion delTMflex_Ferritin (gc) i-3C30-hSL- stabilized (K986P_V987P) N5 protein R9848 Spike pre-S_stab_PP_hex opt1 —/muag; A64-N5- 22732 22759 22813 fusion(K986P_V987P_ (gc) i-3 C30-hSL- stabilized F817P_A892P_ N5 proteinA899P_A942P) R9926 RBD RBD_Foldon opt1 —/muag; A64-N5- 22734 22761 22815fragment (gc) i-3 C30-hSL- (334-528) N5 spike protein R9927 RBDRBD_Foldon opt1 HSD17B4/ hSL-A100 22734 22761 22788 fragment (gc) PSMB3;(334-528) a-1 spike protein R10335 RBD RBD_LumSynth opt1 —/muag; A64-N5-22735 22762 22816 fragment (gc) i-3 C30-hSL- (334-528) N5 spike proteinR10338 RBD RBD_LumSynth opt1 HSD17B4/ hSL-A100 22735 22762 22789fragment (gc) PSMB3; (334-528) a-1 spike protein R10336 RBD LumSynth_RBDopt1 —/muag; A64-N5- 22736 22763 22817 fragment (gc) i-3 C30-hSL-(334-528) N5 spike protein R10339 RBD LumSynth_RBD opt1 HSD17B4/hSL-A100 22736 22763 22790 fragment (gc) PSMB3; (334-528) a-1 spikeprotein R10337 RBD RBD_Ferritin opt1 —/muag; A64-N5- 22733 22760 22814fragment (gc) i-3 C30-hSL- (334-528) N5 spike protein R10340 RBDRBD_Ferritin opt1 HSD17B4/ hSL-A100 22733 22760 22787 fragment (gc)PSMB3; (334-528) a-1 spike protein R10182 S(D614G) S (D614G) opt1HSD17B4/ hSL-A100 22737 22764 22791 (gc) PSMB3; a-1 R10165 Spike pre-S_stab_PP opt1 —/muag; A64-N5- 22738 22765 22819 fusion (K986P_V987P_(gc) i-3 C30-hSL- stabilized D614G) N5 protein R10166 Spike pre-S_stab_PP opt1 HSD17B4/ hSL-A100 22738 22765 22792 fusion (K986P_V987P_(gc) PSMB3; stabilized D614G) a-1 protein R10276 Spike S opt1 —/muag;A64-N5- 22739 22766 22820 protein (A222V_D614G) (gc) i-3 C30-hSL- N5R10278 Spike S opt1 HSD17B4/ hSL-A100 22739 22766 22793 protein(A222V_D614G) (gc) PSMB3; a-1 R10277 Spike pre- S_stab_PP opt1 —/muag;A64-N5- 22740 22767 22821 fusion (K986P_V987P_ (gc) i-3 C30-hSL-stabilized A222V_D614G) N5 protein R10279 Spike pre- S_stab_PP opt1HSD17B4/ hSL-A100 22740 22767 22794 fusion (K986P_V987P_ (gc) PSMB3;stabilized A222V_D614G) a-1 protein R10296 Spike S opt1 —/muag; A64-N5-22741 22768 22822 protein (N439K_D614G) (gc) i-3 C30-hSL- N5 R10298Spike S opt1 HSD17B4/ hSL-A100 22741 22768 22795 protein (N439K_D614G)(gc) PSMB3; a-1 R10297 Spike pre- S_stab_PP opt1 —/muag; A64-N5- 2274222769 22823 fusion (K986P_V987P_ (gc) i-3 C30-hSL- stabilizedN439K_D614G) N5 protein R10299 Spike pre- S_stab_PP opt1 HSD17B4/hSL-A100 22742 22769 22796 fusion (K986P_V987P_ (gc) PSMB3; stabilizedN439K_D614G) a-1 protein R10284 Spike S opt1 —/muag; A64-N5- 22743 2277022824 protein (S477N_D614G) (gc) i-3 C30-hSL- N5 R10287 Spike S opt1HSD17B4/ hSL-A100 22743 22770 22797 protein (S477N_D614G) (gc) PSMB3;a-1 R10285 Spike pre- S_stab_PP opt1 —/muag; A64-N5- 22744 22771 22825fusion (K986P_V987P_ (gc) i-3 C30-hSL- stabilized S477N_D614G) N5protein R10286 Spike pre- S_stab_PP opt1 HSD17B4/ hSL-A100 22744 2277122798 fusion (K986P_V987P_ (gc) PSMB3; stabilized S477N_D614G) a-1protein R10350 Spike S opt1 —/muag; A64-N5- 22745 22772 22826 protein(N501Y_D614G) (gc) i-3 C30-hSL- N5 R10351 Spike pre- S_stab_PP opt1—/muag; A64-N5- 22746 22773 22827 fusion (K986P_V987P_ (gc) i-3 C30-hSL-stabilized N501Y_D614G) N5 protein R10272 Spike S (H69del_V70del_ opt1—/muag; A64-N5- 22747 22774 22828 protein D614G) (gc) i-3 C30-hSL- N5R10274 Spike S (H69del_V70del_ opt1 HSD17B4/ hSL-A100 22747 22774 22801protein D614G) (gc) PSMB3; a-1 R10273 Spike pre- S_stab_PP opt1 —/muag;A64-N5- 22748 22775 22829 fusion (K986P_V987P_ (gc) i-3 C30-hSL-stabilized H69del_V70del_ N5 protein D614G) R10275 Spike pre- S_stab_PPopt1 H5D17B4/ hSL-A100 22748 22775 22802 fusion (K986P_V987P_ (gc)PSMB3; stabilized H69del_V70del_ a-1 protein D614G) R10280 Spike S opt1—/muag; A64-N5- 22749 22776 22830 protein (Y453F_D614G) (gc) i-3C30-hSL- N5 R10282 Spike S opt1 HSD17B4/ hSL-A100 22749 22776 22803protein (Y453F_D614G) (gc) PSMB3; a-1 R10281 Spike pre- S_stab_PP opt1—/muag; A64-N5- 22750 22777 22831 fusion (K986P_V987P_ (gc) i-3 C30-hSL-stabilized Y453F_D614G) N5 protein R10283 Spike pre- S_stab_PP opt1HSD17B4/ hSL-A100 22750 22777 22804 fusion (K986P_V987P_ (gc) PSMB3;stabilized Y453F_D614G) a-1 protein R10288 Spike S (D614G_I692V) opt1—/muag; A64-N5- 22751 22778 22832 protein (gc) i-3 C30-hSL- N5 R10290Spike S (D614G_I692V) opt1 HSD17B4/ hSL-A100 22751 22778 22805 protein(gc) PSMB3; a-1 R10289 Spike pre- S_stab_PP opt1 —/muag; A64-N5- 2275222779 22833 fusion (K986P_V987P_ (gc) i-3 C30-hSL- stabilizedD614G_I692V) N5 protein R10291 Spike pre- S_stab_PP opt1 HSD17B4/hSL-A100 22752 22779 22806 fusion (K986P_V987P_ (gc) PSMB3; stabilizedD614G_I692V) a-1 protein R10344 Spike S opt1 —/muag; A64-N5- 22753 2278022834 protein (D614G_M1229I) (gc) i-3 C30-hSL- N5 R10345 Spike pre-S_stab_PP opt1 —/muag; A64-N5- 22754 22781 22835 fusion (K986P_V987P_(gc) i-3 C30-hSL- stabilized D614G_M1229I) N5 protein R10292 Spike S(H69del_V70del opt1 —/muag; A64-N5- 22755 22782 22836 proteinA222V_Y453F_ (gc) i-3 C30-hSL- S477N_D614G_ N5 I692V) R10294 Spike S(H69del_V70del_ opt1 HSD17B4/ hSL-A100 22755 22782 22809 proteinA222V_Y453F_ (gc) PSMB3; S477N_D614G_ a-1 I692V) R10293 Spike pre-S_stab_PP opt1 —/muag; A64-N5- 22756 22783 22837 fusion (K986P_V987P_(gc) i-3 C30-hSL- stabilized H69del_V70del_ N5 protein A222V_Y453F_S477N_D614G_ I692V) R10295 Spike pre- S_stab_PP opt1 HSD17B4/ hSL-A10022756 22783 22810 fusion (K986P_V987P_ (gc) PSMB3; stabilizedH69del_V70del_ a-1 protein A222V_Y453F_ S477N_D614G_ I692V) R10346 SpikeS (H69del_V70del_ opt1 —/muag; A64-N5- 22757 22784 22838 proteinY453F_D614G_ (gc) i-3 C30-hSL- I692V_M1229I) N5 R10347 Spike pre-S_stab_PP opt1 —/muag; A64-N5- 22758 22785 22839 fusion (K986P_V987P_(gc) i-3 C30-hSL- stabilized H69del_V70del_ N5 protein Y453F_D614G_I692V_M1229I) R10136, Spike pre- S_stab_PP opt1 —/muag; hSL-A100 10 13724397 R10158** fusion (K986P_V987P) (gc) i-3 stabilized protein R10154Spike pre- S_stab_PP opt1 —/muag; A100 10 137 25717 fusion (K986P_V987P)(gc) i-3 stabilized protein R10153 Spike pre- S_stab_PP opt1 H5D17B4/A100 10 137 24837 fusion (K986P_V987P) (gc) PSMB3; stabilized a-1protein R10155 Spike pre- S_stab_PP opt1 Rpl31/ hSL-A100 10 137 23957fusion (K986P_V987P) (gc) RPS9; e-2 stabilized protein R10156 Spike pre-S_stab_PP opt1 Rpl31/ A100 10 137 25277 fusion (K986P_V987P) (gc) RPS9;e-2 stabilized protein R10183 Spike pre- S_stab_PP opt1 Sic7a3/ hSL-A10010 137 23737 fusion (K986P_V987P) (gc) PSMB3; a- stabilized 3 proteinR10184 Spike pre- S_stab_PP opt1 Sic7a3/ A100 10 137 25057 fusion(K986P_V987P) (gc) PSMB3; a- stabilized 3 protein R10300 Spike pre-S_stab_PP opt1 HSD17B4/ hSL-A25 10 137 26925 fusion (K986P_V987P) (gc)PSMB3; stabilized a-1 protein R10301 Spike pre- S_stab_PP opt1 HSD17B4/hSL-A60 10 137 26926 fusion (K986P_V987P) (gc) PSMB3; stabilized a-1protein R10302 Spike pre- S_stab_PP opt1 HSD17B4/ hSL-A80 10 137 26927fusion (K986P_V987P) (gc) PSMB3; stabilized a-1 protein R10303 Spikepre- S_stab_PP opt1 HSD17B4/ hSL-A90 10 137 26928 fusion (K986P_V987P)(gc) PSMB3; stabilized a-1 protein R10304 Spike pre- S_stab_PP opt1HSD17B4/ hSL-A110 10 137 26929 fusion (K986P_V987P) (gc) PSMB3;stabilized a-1 protein R10305 Spike pre- S_stab_PP opt1 H5D17B4/hSL-A120 10 137 26930 fusion (K986P_V987P) (gc) PSMB3; stabilized a-1protein R10306 Spike pre- S_stab_PP opt1 HSD17B4/ hSL-A140 10 137 26931fusion (K986P_V987P) (gc) PSMB3; stabilized a-1 protein R10307 Spikepre- S_stab_PP opt1 HSD17B4/ hSL 10 137 26932 fusion (K986P_V987P) (gc)PSMB3; stabilized a-1 protein R10308 Spike pre- S_stab_PP opt1 —/muag;A50-N5- 10 137 26933 fusion (K986P_V987P) (gc) i-3 C30-hSL- stabilizedN5 protein R10309 Spike pre- S_stab_PP opt1 —/muag; A35-N5- 10 137 26934fusion (K986P_V987P) (gc) i-3 C30-hSL- stabilized N5 protein R10310Spike pre- S_stab_PP opt1 —/muag; A25-N5- 10 137 26935 fusion(K986P_V987P) (gc) i-3 C30-hSL- stabilized N5 protein R10311 Spike pre-S_stab_PP opt1 —/muag; A73-N5- 10 137 26936 fusion (K986P_V987P) (gc)i-3 C30-hSL- stabilized N5 protein R10312 Spike pre- S_stab_PP opt1—/muag; hSL-N5 10 137 26937 fusion (K986P_V987P) (gc) i-3 stabilizedprotein R10162** Spike pre- S_stab_PP opt10 HSD17B4/ hSL-A100 10 146 151fusion (K986P_V987P) (gc PSMB3; stabilized mod) a-1 protein R10352 SpikeS opt1 —/muag; A64-N5- 22941 22981 23201 protein (H69del_V70del_ (gc)i-3 C30-hSL- Y144del_N501Y_ N5 A570D_D614G_ P681H_T716I_ S982A_D1118H)R10356 Spike S opt1 HSD17B4/ hSL-A100 22941 22981 23421 protein(H69del_V70del_ (gc) PSMB3; Y144del_N501Y_ a-1 A570D_D614G_ P681H_T716I_5982A_D1118H) R10353 Spike pre- S_stab_PP opt1 —/muag; A64-N5- 2295923089 23309 fusion (K986P_V987P_ (gc) i-3 C30-hSL- stabilizedH69del_V70del_ N5 protein Y144del_N501Y_ A570D_D614G_ P681H_T716I_S982A_D1118H) R10357 Spike pre- S_stab_PP opt1 HSD17B4/ hSL-A100 2295923089 23529 fusion (K986P_V987P_ (gc) PSMB3; stabilized H69del_V70del_a-1 protein Y144del_N501Y_ A570D_D614G_ P681H_T716I_ S982A_D1118H)R10358 Spike S opt1 —/muag; A64-N5- 22942 22982 23202 protein(K417N_E484K_ (gc) i-3 C30-hSL- N501Y_D614G) N5 R10359 Spike S opt1HSD17B4/ hSL-A100 22942 22982 23422 protein (K417N_E484K_ (gc) PSMB3;N501Y_D614G) a-1 R10360 Spike pre- S_stab_PP opt1 —/muag; A64-N5- 2296023090 23310 fusion (K986P_V987P_ (gc) i-3 C30-hSL- stabilizedK417N_E484K_ N5 protein N501Y_D614G) R10361 Spike pre- S_stab_PP opt1HSD17B4/ hSL-A100 22960 23090 23530 fusion (K986P_V987P_ (gc) PSMB3;stabilized K417N_E484K_ a-1 protein N501Y_D614G) R10379 Spike pre-S_stab_PP opt1 —/muag; A64-N5- 22961 23091 23311 fusion (K986P_V987P_(gc) i-3 C30-hSL- stabilized L18F_D80A_ N5 protein D215G_L242del_A243del_L244del_ R246I_K417N_ E484K_N501Y_ D614G_A701V) R10384 Spikepre- S_stab_PP opt1 H5D17B4/ hSL-A100 22961 23091 23531 fusion(K986P_V987P_ (gc) PSMB3; stabilized L18F_D80A_ a-1 proteinD215G_L242del_ A243del_L244del_ R246I_K417N_ E484K_N501Y_ D614G_A701V)R10378 Spike pre- S_stab_PP opt1 —/muag; A64-N5- 22962 23092 23312fusion (K986P_V987P_ (gc) i-3 C30-hSL- stabilized E484K_D614G) N5protein R10380 Spike pre- S_stab_PP opt1 —/muag; A64-N5- 22963 2309323313 fusion (K986P_V987P_ (gc) i-3 C30-hSL- stabilized L18F_T20N_ N5protein P265_D138Y_ R190S_K417T_ E484K_N501Y_ D614G_H655Y_ T1027I)R10385 Spike pre- S_stab_PP opt1 HSD17B4/ hSL-A100 22963 23093 23533fusion (K986P_V987P_ (gc) PSMB3; stabilized L18F_T2ON_ a-1 proteinP26S_D138Y_ R190S_K417T_ E484K_N501Y_ D614G_H655Y_ T1027I) R10381 Spikepre- S_stab_PP opt1 —/muag; A64-N5- 22964 23094 23314 fusion(K986P_V987P_ (gc) i-3 C30-hSL- stabilized S13I_W152C_ N5 proteinL452R_D614G) *mRNA R10160 and R10161 were produced with 3′OME Clean Cap.**mRNA R10157, R10158, R10159, R10162 were produced withN(1)-methylpseudouridine (m1ψ)

1.4. Preparation of an LNP Formulated mRNA Composition:

LNPs were prepared using cationic lipids, structural lipids, aPEG-lipids, and cholesterol. Lipid solution (in ethanol) was mixed withRNA solution (aqueous buffer) using a microfluidic mixing device.Obtained LNPs were re-buffered in a carbohydrate buffer via dialysis,and up-concentrated to a target concentration using ultracentrifugationtubes. LNP-formulated mRNA was stored at −80° C. prior to use in invitro or in vivo experiments.

Preferably, lipid nanoparticles were prepared and tested according tothe general procedures described in PCT Pub. Nos. WO 2015/199952, WO2017/004143 and WO 2017/075531, the full disclosures of which areincorporated herein by reference. Lipid nanoparticle (LNP)-formulatedmRNA was prepared using an ionizable amino lipid (cationic lipid),phospholipid, cholesterol and a PEGylated lipid. LNPs were prepared asfollows. Cationic lipid according to formula III-3 (ALC-0315), DSPC,cholesterol and PEG-lipid according to formula IVa (ALC-0159) weresolubilized in ethanol at a molar ratio of approximately47.5:10:40.8:1.7 (see Table A). Lipid nanoparticles (LNP) comprisingcompound III-3 were prepared at a ratio of mRNA (sequences see Table 4)to Total Lipid of 0.03-0.04 w/w. Briefly, the mRNA was diluted to 0.05to 0.2 mg/mL in 10 to 50 mM citrate buffer, pH 4. Syringe pumps wereused to mix the ethanolic lipid solution with the mRNA aqueous solutionat a ratio of about 1:5 to 1:3 (vol/vol) with total flow rates above 15ml/min. The ethanol was then removed and the external buffer replacedwith PBS by dialysis. Finally, the lipid nanoparticles were filteredthrough a 0.2 μm pore sterile filter. Lipid nanoparticle particlediameter size was 60-90 nm as determined by quasi-elastic lightscattering using a Malvern Zetasizer Nano (Malvern, UK).

TABLE A Lipid-based carrier composition of the examples Ratio Compounds(mol %) Structure Mass 1 Cholesterol 40.9

386.4 2 1,2-distearoyl- sn-glycero-3- phosphocholine (DSPC) 10

789.6 3 Cationic Lipid 47.4

765.7 4 PEG Lipid 1.7

2010.1

1.5. Preparation of a Protamine Complexed mRNA Composition (Prophetic):

RNA constructs are complexed with protamine prior to use in in vivoimmunization experiments. The RNA formulation consists of a mixture of50% free RNA and 50% RNA complexed with protamine at a weight ratio of2:1. First, mRNA is complexed with protamine by addition ofprotamine-Ringer's lactate solution to mRNA. After incubation for 10minutes, when the complexes are stably generated, free mRNA is added,and the final concentration is adjusted with Ringer's lactate solution.

1.6. Expression Analysis of Designed mRNA Constructs:

The mRNA constructs as shown in Table 4 were tested for their expressionvia in vitro translation using Rabbit Reticulocte Lysate System as wellas in cell culture followed by detection via western blot, or FACSanalysis as commonly known in the art (see for further details andexemplary results Example 2).

Example 2a: Expression Analysis of mRNA Constructs Encoding SARS-CoV-2Proteins (S, S_Stab, S1

To determine in vitro protein expression of the mRNA constructs, theconstructs encoding SARS-CoV-2 Spike proteins or fragments (S, S_stab,S1) were mixed with components of Promega Rabbit Reticulocyte LysateSystem according to manufacturer's protocol. The lysate contains thecellular components necessary for protein synthesis (tRNA, ribosomes,amino acids, initiation, elongation and termination factors). Aspositive control, Luciferase RNA from Lysate System Kit was used. Thetranslation result was analyzed by SDS-Page and Western Blot analysis(IRDye 800CW streptavidin antibody (1:2000)). Table 5 summarizes thetested RNA constructs.

TABLE 5 Overview of mRNA constructs used in Example 2a SEQ Short CDSmRNA ID NO: Lane Name name opt. ID RNA 1 Spike protein S opt1 R9514 162,12743 2 Spike pre-fusion S_stab opt10 R9519 165, 13013 stabilizedprotein 3 Spike pre-fusion S_stab opt1 R9515 163, 12810 stabilizedprotein 4 S fragment (1-681) S1 opt10 R9518 168, 13027 spike protein 5 Sfragment (1-681) S1 opt1 R9513 166, 12824 spike protein 6 RNAse freewater 7 Positive control, control RNA from Lysate System Kit

Results:

As shown in FIG. 1 the used mRNA constructs led to a detectable proteinexpression of the expected size (S or S_stab: 140 kDa, S1: 75 kDa),which is a prerequisite for an mRNA-based SARS-CoV-2 vaccine.

Example 2b: Expression of SARS-CoV-2 Proteins (S, S_Stab, S1) in HeLaCells and Analysis by FACS

To determine in vitro protein expression of the mRNA constructs, HeLacells were transiently transfected with mRNA encoding SARS-CoV-2proteins (S, S_stab, S1) and stained using suitable anti-spike proteinantibodies (raised in mouse), counterstained with a FITC-coupledsecondary antibody. HeLa cells were seeded in a 6-well plate at adensity of 400,000 cells/well in cell culture medium (RPMI, 10% FCS, 1%L-Glutamine, 1% Pen/Strep), 24h prior to transfection. HeLa cells weretransfected with 2 μg unformulated mRNA using Lipofectamine 2000(Invitrogen). The mRNA constructs prepared according to Example 1 andlisted in Table 6 were used in the experiment, including a negativecontrol (water for injection). 24 hours post transfection, HeLa cellswere stained with suitable anti-spike protein (S specific antibodiesraised against SARS S, cross-reactive against SARS-Cov-2 S) antibodies(raised in mouse; 1:250) and anti-mouse FITC labelled secondary antibody(1:500) and subsequently were analyzed by flow cytometry (FACS) on a BDFACS Canto II using the FACS Diva software. Quantitative analysis of thefluorescent FITC signal was performed using the FlowJo software package(Tree Star, Inc.).

TABLE 6 Overview of mRNA constructs used in Example 2b Short CDS mRNASEQ ID Lane Name name opt. ID NO: RNA 1 Spike protein S opt1 R9514 162,12743 2 Spike pre-fusion S_stab opt1 R9515 163, 12810 stabilized protein3 Spike pre-fusion S_stab opt10 R9519 165, 13013 stabilized protein 4 Sfragment (1-681) S1 opt1 R9513 166, 12824 spike protein 5 S fragment(1-681) S1 opt10 R9518 168, 13027 spike protein 6 RNAse free water

Results:

As shown in FIG. 2 the used mRNA constructs led to a detectable cellsurface expression for full length S (S and S_stab). Since the S1fragments lack a transmembrane domain, their expression is notdetectable on the cell surface (FIG. 2).

Example 2c: Expression Analysis of SARS-CoV-2 Proteins (S, S_Stab, S1)Using Western Blot

For the analysis of SARS-CoV-2 S expression, HeLa cells were transfectedwith unformulated mRNA using Lipofectamine as the transfection agent.HeLa cells were seeded in a 6-well plate at a density of 300,000cells/well. HeLa cells were transfected with 2 μg unformulated mRNAusing Lipofectamine 2000 (Invitrogen). The mRNA constructs preparedaccording to Example 1 and listed in Table 7 were used in theexperiment, including a negative control (water for injection). 24 hpost transfection, HeLa cells are detached by trypsin-free/EDTA buffer,harvested, and cell lysates are prepared. Cell lysates were subjected toSDS-PAGE followed by western blot detection. Western blot analysis wasperformed using an anti-spike protein (SARS S, cross-reactive againstSARS-CoV-2 S) antibody used in combination with a suitable secondaryantibody.

TABLE 7 Overview of mRNA constructs used in Example 2c Short CDS mRNASEQ ID Lane Name name opt. ID NO: RNA 1 S fragment (1-681) S1 opt1 R9513166, 12824 spike protein 2 Spike protein S opt1 R9514 162, 12743 3 Sfragment (1-681) S1 opt10 R9518 168, 13027 spike protein 4 Spikepre-fusion S_stab opt10 R9519 165, 13013 stabilized protein 5 Spikepre-fusion S_stab opt1 R9515 163, 12810 stabilized protein 6 RNAse freewater

Results:

Expression was detectable for all analyzed mRNAs in cell lysates (FIG.3), full length S: expected size 140 kDa, two main bands of approx. 90kDA and 180 kDa, likely reflecting glycosylated forms of unprocessed Sprotein (S0) and the cleaved S2 subunit, S1: 70 kDa, likelyglycosylated).

Example 3: Vaccination of Mice with mRNA Encoding SARS-CoV-2 ProteinDesigns Antigens (S, S_Stab

Preparation of LNP Formulated mRNA Vaccine:

SARS-CoV-2mRNA constructs are prepared as described in Example 1 (RNA invitro transcription). HPLC purified mRNA is formulated with LNPsaccording to Example 1.4 prior to use in in vivo vaccinationexperiments.

Immunization:

Female BALB/c mice (6-8 weeks old) are injected intramuscularly (i.m.)with mRNA vaccine compositions and doses as indicated in Table 5. As anegative control, one group of mice is vaccinated with buffer. Allanimals are vaccinated on day 0 and 21. Blood samples are collected onday 21 (post prime) and 42 (post boost) for the determination ofantibody titers.

TABLE 8 Vaccination regimen (Example 3): Group SARS-CoV-2 spike CDS SEQprotein ID NO: optimization Formulation Dose 1 S opt1 LNP 5 μg SEQ IDNO: 148, 155, 162, or 169 2 S opt1 LNP 2.5 μg SEQ ID NO: 148, 155, 162,or 169 3 S opt1 LNP 1.25 μg SEQ ID NO: 148, 155, 162, or 169 4 Sstabilized (S_stab) opt1 LNP 5 μg SEQ ID NO: 149, 156, 163, or 170 5 Sstabilized (S_stab) opt1 LNP 2.5 μg SEQ ID NO: 149, 156, 163, or 170 6 Sstabilized (S_stab) opt1 LNP 1.25 μg SEQ ID NO: 149, 156, 163, or 170 7S stabilized (S_stab) opt3 LNP 5 μg SEQ ID NO: 150, 157, 164, or 171 8 Sstabilized (S_stab) opt3 LNP 2.5 μg SEQ ID NO: 150, 157, 164, or 171 9 Sstabilized (S_stab) opt3 LNP 1.25 μg SEQ ID NO: 150, 157, 164, or 171 7S stabilized (S_stab) opt10 LNP 5 μg SEQ ID NO: 151, 158, 165, or 172 85 stabilized (S_stab) opt10 LNP 2.5 μg SEQ ID NO: 151, 158, 165, or 1729 S stabilized (S_stab) opt10 LNP 1.25 μg SEQ ID NO: 151, 158, 165, or172 10 buffer

Determination of IgG1 and IgG2 Antibody Titers Using ELISA:

ELISA is performed using recombinant SARS-CoV-2 S (extracellular domain)protein for coating. Coated plates are incubated using respective serumdilutions, and binding of specific antibodies to the SARS-CoV-2 S aredetected using biotinylated isotype specific anti-mouse antibodiesfollowed by streptavidin-HRP (horse radish peroxidase) with Amplex assubstrate. Endpoint titers of antibodies (IgG1, IgG2a) are measured byELISA on day 21, and 42 post vaccinations.

Detection of Spike Protein-Specific Immune Responses:

Hela cells are transfected with 2 μg of mRNA encoding spike proteinusing lipofectamine. The cells are harvested 20h post transfection, andseeded at 1×10⁶ per well into a 96 well plate. The cells are incubatedwith serum samples of vaccinated mice (diluted 1:50) followed by aFITC-conjugated anti-mouse IgG antibody. Cells were acquired on BD FACSCanto II using DIVA software and analyzed by FlowJo.

Intracellular Cytokine Staining:

Splenocytes from vaccinated mice are isolated according to a standardprotocol known in the art. Briefly, isolated spleens are grinded througha cell strainer and washed in PBS/1% FBS followed by red blood celllysis. After an extensive washing step with PBS/1% FBS, splenocytes areseeded into 96-well plates (2×10⁶ cells per well). Cells are stimulatedwith a mixture of SARS-CoV-2 S protein specific peptide epitopes (5μg/ml of each peptide) in the presence of 2.5 μg/ml of an anti-CD28antibody (BD Biosciences) for 6 hours at 37° C. in the presence of aprotein transport inhibitor. After stimulation, cells are washed andstained for intracellular cytokines using the Cytofix/Cytoperm reagent(BD Biosciences) according to the manufacturer's instructions. Thefollowing antibodies are used for staining: Thy1.2-FITC (1:200),CD8-APC-H7 (1:100), TNF-PE (1:100), IFNγ-APC (1:100) (eBioscience),CD4-BD Horizon V450 (1:200) (BD Biosciences) and incubated withFcγ-block diluted 1:100. Aqua Dye is used to distinguish live/dead cells(Invitrogen). Cells are acquired using a Canto II flow cytometer(Beckton Dickinson). Flow cytometry data is analyzed using FlowJosoftware package (Tree Star, Inc.)

Determination of Virus Neutralization Titers:

Serum is collected for determination of SARS-CoV-2 neutralization titers(VNTs) detected via CPE (cytopathic effect) or via a pseudotypedparticle-based assay.

Example 4: Vaccination of Mice with mRNA Encoding SARS-CoV-2 Antigen S1

Preparation of LNP Formulated mRNA Vaccine:

SARS-CoV-2 mRNA constructs are prepared as described in Example 1 (RNAin vitro transcription). HPLC purified mRNA is formulated with LNPsaccording to Example 1.4 prior to use in in vivo vaccinationexperiments.

Immunization:

Female BALB/c mice (6-8 weeks old) are injected intramuscularly (i.m.)with mRNA vaccine compositions and doses as indicated in Table 6. As anegative control, one group of mice is vaccinated with buffer. Allanimals are vaccinated on day 0 and 21. Blood samples are collected onday 21 (post prime) and 42 (post boost) for the determination ofantibody titers.

TABLE 9 Vaccination regimen (Example 4) SARS-CoV-2 spike CDS Groupprotein SEQ ID NO: optimization Formulation Dose 1 Spike S1 opt1 LNP 5μg SEQ ID NO: 152, 159, 166, or 173 2 Spike S1 opt1 LNP 2.5 μg SEQ IDNO: 152, 159, 166, or 173 3 Spike S1 opt1 LNP 1.2 μg SEQ ID NO: 152,159, 166, or 173 1 Spike S1 opt3 LNP 5 μg SEQ ID NO: 153, 160, 167, or161 2 Spike S1 opt3 LNP 2.5 μg SEQ ID NO: 153, 160, 167, or 161 3 SpikeS1 opt3 LNP 1.25 μg SEQ ID NO: 153, 160, 167, or 161 1 Spike S1 opt10LNP 5 μg SEQ ID NO: 154, 161, 168, or 175 2 Spike S1 opt10 LNP 2.5 μgSEQ ID NO: 154, 161, 168, or 175 3 Spike S1 opt10 LNP 1.25 μg SEQ ID NO:154, 161, 168, or 175 4 buffer

The induction of specific immune responses via ELISA, ICS and VNTs aredetermined as described before (see Example 3).

Example 5: Vaccination of Mice with mRNA Encoding SARS-CoV-2 AntigenDesign (S_Stab

Preparation of LNP Formulated mRNA Vaccine:

SARS-CoV-2 S mRNA constructs are prepared as described in Example 1 (RNAin vitro transcription). HPLC purified mRNA was formulated with LNPsaccording to Example 1.4.

Immunization:

Female BALB/c mice (6-8 weeks old) were injected intramuscularly (i.m.)with mRNA vaccine compositions and doses as indicated in Table 10. As anegative control, one group of mice was vaccinated with buffer. Allanimals were vaccinated on day 0. Blood samples were collected on day 21for the determination of antibody titers.

TABLE 10 Vaccination regimen (Example 5): SEQ SEQ Vaccine mRNA CDS IDNO: ID NO: Group composition ID opt. Protein RNA Dose A mRNA encodingR9519 opt10 10, 341 165, 13013   2 μg S_stab formulated in LNPs B Rec.protein — — 1.5 μg SARS-CoV-2 S ECD (extracellular domain) + alum Cbuffer — — —

Determination of IgG1 and IgG2 Antibody Titers Using ELISA:

ELISA was performed using recombinant SARS-CoV-2 S protein for coating.Coated plates were incubated using respective serum dilutions, andbinding of specific antibodies to SARS-CoV-2 S were detected usingbiotinylated isotype specific anti-mouse antibodies followed bystreptavidin-HRP (horse radish peroxidase) with Amplex as substrate.Endpoint titers of antibodies (IgG1, IgG2a) were measured by ELISA onday 21, after one single prime vaccination.

Results:

As shown in FIG. 4 the vaccination with mRNA encoding for full length Sstabilized protein induced high titers of S specific binding antibodyafter a single vaccination (d21). Compared to the adjuvanted recombinantS protein the mRNA vaccine induced comparable IgG1 titers and higherIgG2a titers.

Example 6: Vaccination of Mice with mRNA Encoding SARS-CoV-2 AntigenDesigns

Preparation of LNP Formulated mRNA Vaccine:

SARS-CoV-2 S mRNA constructs are prepared as described in Example 1 (RNAin vitro transcription). HPLC purified mRNA was formulated with LNPsaccording to Example 1.4.

Immunization:

Female BALB/c mice (6-8 weeks old) were injected intramuscularly (i.m.)with mRNA vaccine compositions and doses as indicated in Table 11. As anegative control, one group of mice was vaccinated with buffer. Allanimals were vaccinated on day 0 and 21. Blood samples were collected onday 21 (post prime) and 42 (post boost) for the determination ofantibody titers.

TABLE 11 Vaccination regimen (Example 6): SEQ SEQ mRNA CDS ID NO: ID NO:Group Vaccine composition ID opt. Protein RNA Dose A mRNA encodingS_full R9514 opt1  1 162 2 μg length formulated in LNPs B mRNA encodingS_stab R9515 opt1 10 163 2 μg formulated in LNPs C mRNA encoding S_stabR9519 opt10 10 165 2 μg formulated in LNPs D buffer — — —

Determination of IgG1 and IgG2 Antibody Titers Using ELISA:

ELISA was performed using recombinant SARS-CoV-2 S (extracellulardomain) protein for coating. Coated plates were incubated usingrespective serum dilutions, and binding of specific antibodies toSARS-CoV-2 S were detected using biotinylated isotype specificanti-mouse antibodies followed by streptavidin-HRP (horse radishperoxidase) with Amplex as substrate. Endpoint titers of antibodies(IgG1, IgG2a) were measured by ELISA on day 21, and 42 post primevaccination.

Determination of Virus Neutralization Titers:

Serum was collected for determination of SARS-CoV-2 neutralizationtiters (VNTs) detected via CPE (cytopathic effect). Serial dilutions ofheat-inactivated sera (56° C. for 30 min) tested in duplicates with astarting dilution of 1:10 followed by 1:2 serial dilutions wereincubated with 100 TCID₅₀ of wild type SARS-CoV-2 (strain2019-nCov/Italy-INMI1) for 1 hour at 37° C. Every plate contained adedicated row (8 wells) for cell control which contains only cells andmedium, and a dedicated row of virus control which contain only cellsand virus. Infectious virus was quantified upon incubation of 100 μl ofvirus-serum mixture with a confluent layer of Vero E6 cells (ATCC, Cat.1586) followed by incubation for 3 days at 37° C. and microscopicalscoring for CPE formation. A back titration was performed for each runin order to verify the correct range of TCID50 of the working virussolution. VN titres were calculated according to the method described byReed & Muench. If no neutralization was observed (MNt<10), an arbitraryvalue of 5 was reported. Analyses were carried out at VisMederi srl(Siena, Italy).

Intracellular Cytokine Staining:

Splenocytes from vaccinated mice were isolated according to a standardprotocol known in the art. Briefly, isolated spleens were grindedthrough a cell strainer and washed in PBS/1% FBS followed by red bloodcell lysis. After an extensive washing step with PBS/1% FBS, splenocyteswere seeded into 96-well plates (2×10⁶ cells per well). Cells werestimulated with a mixture of SARS-CoV-2 S protein specific peptideepitopes (5 μg/ml of each peptide) in the presence of 2.5 μg/ml of ananti-CD28 antibody (BD Biosciences) for 6 hours at 37° C. in thepresence of a protein transport inhibitor. After stimulation, cells werewashed and stained for intracellular cytokines using theCytofix/Cytoperm reagent (BD Biosciences) according to themanufacturer's instructions. The following antibodies were used forstaining: Thy1.2-FITC (1:200), CD8-APC-H7 (1:100), TNF-PE (1:100),IFNγ-APC (1:100) (eBioscience), CD4-BD Horizon V450 (1:200) (BDBiosciences) and incubated with Fcγ-block diluted 1:100. Aqua Dye wasused to distinguish live/dead cells (Invitrogen). Cells were acquiredusing a Canto II flow cytometer (Beckton Dickinson). Flow cytometry datawas analyzed using FlowJo software package (Tree Star, Inc.)

Results:

As shown in FIGS. 5A and B the vaccination with mRNA encoding fulllength S protein and full length S stabilized protein (S_stab) inducedhigh titers of S specific binding antibody after a single vaccination(d21) (FIG. 5A: IgG1, FIG. 5B: IgG2a). The titers increased after asecond vaccination (d42). All mRNA designs induced more or lesscomparable antibody titers, whereas mice of group C showed a decreasedlevel of IgG2a antibodies on d21 compared to other groups. As shown inFIG. 5C the vaccination with mRNA encoding for full length S protein andfull length S stabilized protein induced robust levels of virusneutralizing antibodies after two vaccinations.

As shown in FIG. 6 the vaccination with mRNA encoding for full length Sprotein and full length S stabilized (S_stab) protein induced both CD4⁺and CD8⁺ IFNγ/TNF double positive T cells.

Example 7: In Vivo Immunogenicity of SARS-CoV-2 Vaccine CompositionFollowing Different Vaccination Schedules

Preparation of LNP Formulated mRNA Vaccine:

SARS-CoV-2 S mRNA constructs are prepared as described in Example 1 (RNAin vitro transcription). HPLC purified mRNA were formulated with LNPsaccording to Example 1.4.

Immunization:

Female BALB/c mice (6-8 weeks old) were injected intramuscularly (i.m.)with mRNA vaccine compositions and doses as indicated in Table 12. GroupI was vaccinated with Alum adjuvanted SARS-CoV2 spike protein (Sextracellular domain) (1.5 μg in 5.6 μl Alhydrogel buffered in phosphatebuffered saline [PBS]). As a negative control, one group of mice wasvaccinated with buffer (0.9% NaCl).

Animals received their first vaccination on day 0, day 7, day 14 or day21. All animals received a second vaccination on day 28. The presence ofSARS-CoV 2 S binding antibodies was analyzed on day 28 and day 35, thepresence of virus-neutralizing titers (VNTs) was analyzed on day 28, 35and 49. The induction of T cell responses after vaccination was assessedon day 49 of the experiment. This experimental setup was chosen todetermine the onset of the specific immune responses and to ascertainwhich vaccination interval from first to second immunization yields thehighest immune responses in mice.

TABLE 12 Vaccination regimen (Example 7). mRNA CDS SEQ ID SEQ ID GroupVaccine composition ID vaccination opt. NO: Protein NO: RNA Dose A mRNAencoding S_stab R9515 d21, d28 opt1 10 163 2 μg formulated in LNPs(CVnCoV) B mRNA encoding S_stab R9515 d14, d28 opt1 10 163 2 μgformulated in LNPs (CVnCoV) C mRNA encoding S_stab R9515  d7, d28 opt110 163 2 μg formulated in LNPs (CVnCoV) D mRNA encoding S_stab R9515 d0, d28 opt1 10 163 2 μg formulated in LNPs (CVnCoV) E mRNA encodingS_stab R9519 d21, d28 opt10 10 165 2 μg formulated in LNPs F mRNAencoding S_stab R9519 d14, d28 opt10 10 165 2 μg formulated in LNPs GmRNA encoding S_stab R9519  d7, d28 opt10 10 165 2 μg formulated in LNPsH mRNA encoding S_stab R9519  d0, d28 opt10 10 165 2 μg formulated inLNPs I Pos. control (alum —  d0, d28 — — — — adjuvanted S protein) JBuffer —  d0, d28 — — — —

Characterisation of RNA-Induced Innate Immune Responses:

Blood samples were taken via retro-orbital bleeding 14h afteradministration of mRNA encoding S_stab formulated in LNPs (exemplarilyshown for group A), positive control, or buffer. Serum cytokines (IFN-γ,IL-1β TNF, IL-6, IL-4, IL-5 and IL-13) were assessed using cytometricbead array (CBA) using the BD FACS CANTO II. Serum was diluted 1:4 andBD Bioscience mouse cytokine flex sets were used according tomanufacturer's protocol to determine serum cytokine levels.

The following flex set were used: Mouse IFN-γ Flex Set RUO (A4) (BDBioscience, Cat. 558296); Mouse 11-13 Flex Set RUO (B8) (BD Bioscience,Cat. 558349); Mouse IL-1β Flex Set RUO (E5) (BD Bioscience, Cat.560232); Mouse 11-4 Flex Set RUO (A7) (BD Bioscience, Cat. 558298);Mouse 11-5 Flex Set RUO (A6) (BD Bioscience, Cat. 558302); Mouse IL-6Flex Set RUO (B4) (BD Bioscience, Cat. 558301); Mouse TNF Flex Set RUO(C8) (BD Bioscience, Cat. 558299). IFN-α was quantified usingVeriKine-HS Mouse IFN-α Serum ELISA Kit (01, Cat. 42115-1) according tomanufacturer's instructions. Sera were diluted 1:100 and 50 μl of thedilution was tested.

Determination of IgG1 and IgG2 Antibody Titers Using ELISA:

ELISA was performed using recombinant SARS-CoV-2 S (extracellulardomain) protein for coating. Coated plates were incubated usingrespective serum dilutions, and binding of specific antibodies toSARS-CoV-2 S were detected using biotinylated isotype specificanti-mouse antibodies followed by streptavidin-HRP (horse radishperoxidase) with Amplex as substrate.

Determination of Virus Neutralization Titers:

Serum was collected for determination of SARS-CoV-2 neutralizationtiters (VNTs) detected via CPE (cytopathic effect) using wild typeSARS-CoV-2 virus. For the analysis of virus neutralizing titres of mousesera, serial dilutions of heat-inactivated sera (56° C. for 30 min)tested in duplicates with a starting dilution of 1:10 followed by 1:2serial dilutions were incubated with 100 TCID₅₀ of wild type SARS-CoV-2(strain 2019-nCov/Italy-INMI1) for 1 hour at 37° C. Every platecontained a dedicated row (8 wells) for cell control which contains onlycells and medium, and a dedicated row of virus control which containonly cells and virus. Infectious virus was quantified upon incubation of100 μl of virus-serum mixture with a confluent layer of Vero E6 cells(ATCC, Cat. 1586) followed by incubation for 3 days at 37° C. andmicroscopical scoring for CPE formation. A back titration was performedfor each run in order to verify the correct range of TCID₅₀ of theworking virus solution. VN titres were calculated according to themethod described by Reed & Muench. If no neutralization was observed(MNt<10), an arbitrary value of 5 was reported. Analyses were carriedout at VisMederi srl (Siena, Italy).

Intracellular Cytokine Staining:

Splenocytes were isolated and stimulated with SARS-CoV-2 spike specificpeptide library for 24 hours. Subsequently cells were stained forcluster of differentiation 8 (CD8) and CD4 T cells (surface) and forINF-γ and TNF (intracellular) to evaluate the induction ofmultifunctional T cells specifically activated by vaccine-specificpeptides. Cells incubated with dimethyl sulfoxide (DMSO) served asnegative controls. Cells were acquired using a Canto II flow cytometer(Beckton Dickinson). Flow cytometry data was analyzed using FlowJosoftware package (Tree Star, Inc.)

Results

As shown in FIG. 7 the cytokine analyses demonstrated the induction of abalanced immune response upon mRNA encoding S_stab formulated in LNPs(CVnCoV) injection that exhibited no bias towards IFNγ or IL4, IL-5 andIL-13, indicative of a TH1 and TH2 response, respectively. Low levels ofpro-inflammatory cytokines IL-6, IFNα were detectable in serum, whileTNF and IL1β remained undetectable.

As shown in FIG. 8A the vaccination with mRNA R9515 encoding full lengthS stabilized protein (S_stab) induced a fast onset of immune responseupon first vaccination. A single i.m. administration of the vaccinecomposition was sufficient to induce binding antibodies seven dayspost-injection.

As shown in FIG. 8B the vaccination with mRNA encoding full length Sstabilized protein (S_stab) induced comparable antibody titersindependently of CDS optimization. Levels of binding antibodiesincreased with longer intervals between vaccination and serum sampling(FIG. 8A+B). A second immunization was able to increase the overalltiters of binding antibodies one week post-injection (day 35). Higherlevels of binding antibody titers were observed on day 35 in groupsfeaturing longer intervals between first and second immunization.Adjuvanted recombinant spike protein vaccine (group I) inducedcomparable levels of binding IgG1 antibodies, but IgG2a titers werestatistically significantly lower compared to all mRNA groups.

As shown in FIG. 9A+B, low, but detectable levels of VNTs were present28 days post first vaccination (group D and H). VNT levels increasedafter the second immunization across all groups analyzed on day 35 andday 49 of the study. In line with the increased binding antibodies, VNTsincreased over time and for groups with longer intervals between firstand second vaccination.

As shown in FIG. 10, a strong increase in multifunctional CD8+ and CD4+T cells was observed in vaccinated animals.

Strong induction of multifunctional T cells as well as binding and, moreimportantly, of functional antibodies suggest that the mRNA vaccineencoding the SARS-CoV-2 spike protein elicits potent immune responses inmice.

The vaccine elicited a balanced Th1/Th2 profile, indicated by theinduction of comparable levels of IgG1 and IgG2a antibodies as well as acytokine profile that gives no indication of a TH2 bias, i.e. inductionof IL4, IL5 and IL13.

Example 8: Vaccination of Rats with mRNA Encoding SARS-CoV-2 Antigen

Preparation of LNP Formulated mRNA Vaccine:

SARS-CoV-2 S mRNA constructs are prepared as described in Example 1 (RNAin vitro transcription). HPLC purified mRNA was formulated with LNPsaccording to Example 1.4 prior to use in in vivo vaccinationexperiments.

Immunization:

Rats were injected intramuscularly (i.m.) with mRNA vaccine compositionsand doses as indicated in Table 13. As a negative control, one group ofrats was vaccinated with buffer (group A). All animals were vaccinatedon day 0 and day 21. Blood samples were collected on day 21 (post prime)and 42 (post boost) for the determination of antibody titers.

TABLE 13 Vaccination regimen (Example 8): SEQ SEQ mRNA CDS ID NO: ID NO:Group Vaccine composition ID opt. Protein RNA Dose A buffer — — — — BmRNA encoding S_stab R9515 opt1 10 163 0.5 μg formulated in LNPs C mRNAencoding S_stab R9515 opt1 10 163   2 μg formulated in LNPs D mRNAencoding S_stab R9515 opt1 10 163  10 μg formulated in LNPs E mRNAencoding S_stab R9515 opt1 10 163  40 μg formulated in LNPs F mRNAencoding S_stab R9515 opt1 10 163  80 μg formulated in LNPs

Determination of IgG1 and IgG2 Antibody Titers Using ELISA:

ELISA was performed using recombinant SARS-CoV-2 S (extracellulardomain) protein for coating. Coated plates were incubated usingrespective serum dilutions, and binding of specific antibodies toSARS-CoV-2 S were detected directly with labeled HRP antibody instead ofa secondary HRP antibody used for mouse ELISA. The lack of signalamplification in rat ELISA might account for lower titers, thereforeELISA titers between rat and mouse studies are currently not comparable.

Determination of Virus Neutralizing Antibody Titers (VNT)

Virus neutralizing antibody titers (VNT) of rat serum samples wereanalyzed as previously described in Example 6 with mouse serum.

Results:

As shown in FIG. 11A-C the vaccination with mRNA full length Sstabilized protein formulated in LNPs induced in rats dose dependentlevels of binding antibody titers at day 21 using doses of 0.5 μg, 2 μgand 10 μg and reached saturation in groups vaccinated with 40 μg and 80μg. FIGS. 11D and E show levels of binding antibody titers at day 42after the first vaccination. The second vaccination led to a furtherincrease of antibody titers.

As shown in FIGS. 11F and G the vaccination with mRNA full length Sstabilized protein formulated in LNPs induced in rats dose dependentlevels of VNTs.

Example 9: Challenge Study of Hamsters with SARS-CoV-2

The protective efficacy of mRNA encoding S_stab formulated in LNPs(CVnCoV) was addressed in Syrian hamsters. This model represents mild tomoderate human lung disease pathology and is one of the recognized andaccepted models to investigate human-relevant immunogenicity andpathogenesis (Munoz-Fontela et al, PMID 32967005). Hamsters aresusceptible to wild-type SARS-CoV-2 infection, resulting in high levelsof virus replication and histopathological changes in viral targetorgans.

Preparation of LNP Formulated mRNA Vaccine:

SARS-CoV-2 S mRNA construct was prepared as described in Example 1 (RNAin vitro transcription). HPLC purified mRNA was formulated with LNPsaccording to Example 1.4 prior to use in in vivo vaccinationexperiments.

Immunization and Challenge:

Syrian golden hamsters (n=5/group, 11 to 13 weeks old) were injectedintramuscularly (i.m.) with mRNA vaccine compositions and doses asindicated in Table 14 (see e.g. group E and F). As negative controls,one group of hamsters was not treated and mock infected (with buffer)(group A), another group was injected with NaCl as a buffer control. Asa positive control, group C was infected intranasally with 10²TCID50/dose of SARS CoV-2 isolate BetaCoV/Munich/BavPat1/2020(containing a D614G substitution) in 0.1 ml on day 0. As an additionalpositive control, group D was injected intramuscularly with 5 μg ofrecombinant SARS-CoV-2 spike protein (S1+S2 ECD, His tag; SinoBiological, Cat. 40589-V08B1) adjuvanted in Alhydrogel (Brenntag) 2%.Blood samples were collected on day 28 (post prime) and day 42 and 56(post boost) for the determination of antibody titers. The animals werechallenged intranasally with 10² TCID50/dose of SARS CoV-2 in a totaldose volume of 0.1 ml at day 56. Animals were followed for four dayspost challenge (p.c.) and euthanised on day 60 of the experiment.

TABLE 14 Vaccination regimen (Example 9). SEQ SEQ mRNA CDS ID NO: ID NO:Group Vaccine composition ID dose vaccination opt. Protein RNA AUntreated/mock infected B NaCl d0, d28 — — — C SARS-CoV-2 infected 10²d0 — — — TCID₅₀ D Pos. control (alum 1.5 μg d0, d28 — — — adjuvanted Sprotein) E mRNA encoding S_stab R9515   2 μg d0, d28 opt1 10 163formulated in LNPs (CVnCoV) F mRNA encoding S_stab R9515  10 μg d0, d28opt1 10 163 formulated in LNPs (CVnCoV)

Antibody Analysis Blood samples were taken at day 28, 42, and 56 for thedetermination of total IgG antibodies via ELISA. Plates were coated with1 μg/ml of SARS-CoV-2 spike S (extracellular domain) protein for 4-5h at37° C. Plates were blocked overnight in 10% milk, washed and incubatedwith serum for 2h at room temperature. For detection, hamster sera wereincubated with biotin goat anti-hamster (Syrian) IgG antibody(BioLegend, Cat: 405601) followed by incubation with HRP-Streptavidin(BD, Cat: 554066). Detection of specific signals was performed in aBioTek SynergyHTX plate reader, with excitation 530/25, emissiondetection 590/35 and a sensitivity of 45. IgG antibody titers via ELISAfor infected animals (group C) were not analyzed.

Virus neutralizing antibody titers (VNT) of hamster serum samples wereanalysed upon heat inactivation of samples for 30 min at 56° C.Triplicate, serial two-fold dilutions were incubated with 10²TCID50/well SARS-CoV-2 virus (featuring the mutation D614G) for one hourat 37° C. leading to a sample starting dilution of 1:10. The virus-serummixtures were transferred to 96 well plates with Vero E6 cell culturemonolayers and incubated for five days at 37° C. Plates were then scoredusing the vitality marker WST8 and (100% endpoint) VN titers werecalculated according to the method described by Reed & Muench.

Viral Load in the Respiratory Tract

Detectable levels of replication competent virus in throat swabs, lungand nasal turbinate tissues post challenge were analysed. Quadruplicate,10-fold serial dilutions were transferred to 96 well plates with Vero E6cell culture monolayers and incubated for one hour at 37° C. Cellmonolayers were washed prior to incubation for five days at 37° C.Plates were then scored using the vitality marker WST8 and viral titers(Log10 TCID50/ml or /g) were calculated using the method ofSpearman-Karber.

Histopathology Upon Challenge in Hamsters

Histopathological analysis was performed on tissues sampled on day 4post challenge. After fixation with 10% formalin, sections were embeddedin paraffin and the tissue sections were stained with haematoxylin andeosin for histological examination. Histopathological assessment scoringis as follows: Alveolitis severity, bronchitis/bronchiolitis severity:0=no inflammatory cells, 1=few inflammatory cells, 2=moderate number ofinflammatory cells, 3=many inflammatory cells. Alveolitis extent, 0=0%,1=<25%, 2=25-50%, 3=>50%. Alveolar oedema presence, alveolar haemorrhagepresence, type II pneumocyte hyperplasia presence, 0=no, 1=yes. Extentof peribronchial/perivascular cuffing, 0=none, 1=1-2 cells thick, 2=3-10cells thick, 3=>10 cells thick.

Results

As shown in FIG. 12A hamsters vaccinated with two CVnCoV doses of 2 μgor 10 μg in a 4-week interval developed dose-dependent S binding IgGantibodies after the first vaccination that increased upon the second.Median endpoint titres of animals vaccinated with 10 μg of CVnCoV were1.6×10⁵ after one dose and peaked at 7.8×10⁵ on day 42. IgG antibodytiters via ELISA for infected animals (group C) were not analyzed.

As shown in FIG. 12B, detectable levels of VNTs were present 28 dayspost first vaccination. VNT levels increased after the secondimmunization across both dose groups (group E and F) analyzed on day 42and day 56 of the study. Virus employed for this assay featured theD614D mutation, while CVnCoV encoded S_stab does not include thismutation. Of note, a control group that received Alum-adjuvanted SECDprotein developed IgG antibodies without inducing detectable levels ofVNTs.

On day 56, four weeks after second vaccination, all animals werechallenged with SARS-CoV-2 featuring D614G (10² TCID₅₀/dose). In buffercontrol animals, levels of replication-competent virus from throatswaps, taken daily from day 56 to termination on day 60, showed peakviral titres of approximately 10³ TCID50/ml two days post challenge thatreturned to nearly undetectable levels on day 60. Animals previouslyinfected with SARS-CoV-2 remained negative throughout the experiment.Viral levels were significantly reduced in throat swabs of both CVnCoVvaccinated groups. Vaccination with 10 μg of CVnCoV resulted insignificantly diminished and delayed viral peaks at 10¹⁵ TCID50/ml threedays post challenge. At least 2 out of 5 animals in this group remainednegative throughout the testing period (see FIG. 12C).

Viral levels in nasal turbinates revealed less pronounced, butdetectable dose-dependent reduction of viral replication (FIG. 12D).Importantly, animals vaccinated with 10 μg of CVnCoV exhibited nodetectable viral levels in the lungs, proving the ability of CVnCoV toprotect animals from viral replication in the lower respiratory tract(FIG. 12E).

Histopathological analyses demonstrated the occurrence of alveolardamage and inflammation of alveoli, bronchi and trachea in the buffercontrol group upon SARS-CoV-2 infection. Consistent with protection fromviral replication in the lungs, CVnCoV significantly reducedhistopathological changes upon two vaccinations with 10 μg. Importantly,a dose of 2 μg, which lead to the induction of binding antibodies butonly elicited low levels of VNTs, did not induce increasedhistopathology scores. Group comparisons for differential geneexpression in lung homogenates showed no significant change in theinduction of IL-4 or IL-5 in the mRNA groups compared to buffer or mockinfection groups (data not shown). Therefore, the inventors concludethat CVnCoV does not induce enhanced disease in hamsters, (e.g. viaantibody dependent enhancement) even under conditions where breakthroughviral replication occurs. The presented data indicates that vaccinationwith Alum-adjuvanted protein vaccine, that elicits no detectable levelsof VNTs but high levels of binding antibodies, causes increasedhistopathology scores in hamsters (FIG. 12F, Table 14).

TABLE 15 List of histopathological analysis indicated in FIG. 12F:Histopathological analysis 1 Extend of alveolitis/alveolar damage 2Severity of alveolitis 3 Sum of extend + severity alveolitis 4 Alveolaroedema presence 5 Alevolar haemorrhage presence 6 Type IIpneumocytehyperplasia presence 7 Severity of bronchitis 8 Severity ofbronchiolitis 9 Degree of peribronchial/perivascular cuffing 10 Severityof tracheitis 11 Severity of rhinitis

Consistent with robust immune responses, CVnCoV protected hamsters fromSARS-CoV-2 viral challenge featuring the D614G mutation in S, provingCVnCoV's ability to protect against the most prevalent form of thevirus. These experiments showed significant reduction in replicatingvirus levels in the upper respiratory tract and the absence ofdetectable live virus in the lungs of animals upon two vaccinations with10 μg of CVnCoV.

Example 10: Clinical Development of a SARS-CoV-2 mRNA VaccineComposition

To demonstrate safety and immunogenicity of the mRNA vaccinecomposition(s), clinical trials (phase 1, 2a) were initiated. In thephase 1 clinical trial, cohorts of human volunteers (18-60 years) wereintramuscularly injected for at least two times (e.g. day 1 and day 29with a dose of 2 μg, 4 μg, 8 μg, 12 μg, 16 μg, or 20 μg mRNA encodingSARS-CoV-2 spike protein (R9515, SEQ ID No. 163) formulated in LNPs (asdescribed in Example 1.4) according to the invention (CVnCoV)). In orderto assess the safety profile of the vaccine compositions according tothe invention, subjects are monitored after administration (vital signs,vaccination site and systemic reactogenicity assessments, hematologicanalysis). The immunogenicity of the immunization is analyzed bydetermination of antibodies against SARS-CoV-2, virus neutralizingtiters (VNT) and SARS-CoV-2 specific T cells in sera from vaccinatedsubjects. Blood samples are collected on day 1 as baseline, after eachvaccination and during long-term follow-up.

Cohorts:

2 μg 4 μg 6 μg 8 μg 12 μg 16 μg 20 μg Total N Sero- 46 46 46 46 24 12 12232 negatives N Sero- 10 10 10 6 4 1 41 positives Total CVnCoV + 56 5656 52 28 13 12 273 N placebo

On Day 8, Day 15, Day 29, Day 36, Day 43, Day 57, Day 120, Day 211 andDay 393 the following was determined:

-   -   a.) The proportion of subjects seroconverting for SARS-CoV-2        spike protein antibodies, as measured by ELISA. In subjects who        did not get exposed to SARS-CoV-2 before the trial, or during        the trial before the applicable sample was collected, as        measured by ELISA to SARS-CoV-2 N-antigen, seroconversion is        defined as an increase in titer in antibodies against SARS-CoV-2        spike protein versus baseline.    -   In subjects seropositive for SARS-CoV-2 at baseline,        seroconversion is defined as a 2-fold increase in titer in        antibodies against SARS-CoV-2 spike protein versus baseline.    -   b.) Individual SARS-CoV-2 spike protein-specific antibody levels        in serum, as measured by ELISA.    -   c.) Geometric mean titers (GMTs) of serum SARS-CoV-2 spike        protein antibodies, as measured by ELISA, in subjects who did        not get exposed to SARS-CoV-2 before the trial or during the        trial before the applicable sample was collected, as measured by        ELISA to SARS-CoV-2 N-antigen.    -   d.) The proportion of subjects seroconverting for SARS-CoV-2        neutralizing antibodies, as measured by an activity assay.    -   In subjects who did not get exposed to SARS-CoV-2 before the        trial or during the trial before the applicable sample was        collected, as measured by ELISA to SARS-CoV-2 N-antigen,        seroconversion is defined as an increase in titer in SARS-CoV-2        neutralizing antibodies versus baseline.    -   In subjects seropositive for SARS-CoV-2 at baseline,        seroconversion is defined as a 2-fold increase in titer in        SARS-CoV-2 neutralizing antibodies versus baseline.    -   e.) Individual SARS-CoV-2 neutralizing antibody levels in serum.    -   f.) GMTs of serum SARS-CoV-2 neutralizing antibodies, as        measured by an activity assay, in subjects who did not get        exposed to SARS-CoV-2 before the trial or during the trial        before the applicable sample was collected, as measured by ELISA        to SARS-CoV-2 N-antigen.

Cell-Mediated Immune Response

On Day 29, Day 36 and Day 211 in peripheral blood mononuclear cells(PBMCs) from all subjects at the assigned site(s) the following wasdetermined:

-   -   a.) The frequency and functionality of SARS-CoV-2 spike-specific        T-cell response after antigen stimulation.    -   b.) Intracellular cytokine staining (ICS) to investigate Th1        response and production of Th2 markers    -   c.) The proportion of subjects with a detectable increase in        SARS-CoV-2 spike-specific T-cell response.

Innate Immune Response

On Day 2, Day 8, Day 29, Day 30 and Day 36 in all open-label sentinelsubjects the following was determined:

-   -   a.) Serum cytokine concentrations, including but not limited to        interferon (IFN)-α, IFN-γ, interleukin (IL)-6, chemokine        ligand (CCL) 2 and IFN-γ-induced protein 10 (IP 10).    -   b.) Gene expression profiling.

Evaluation of Infection

-   -   a.) Number of subjects with virologically-confirmed SARS-CoV-2        infection as measured by reverse transcription (RT)-PCR at        clinically was determined time points throughout the trial.    -   b.) Number of subjects with asymptomatic SARS-CoV-2 infection as        measured by retrospective serology at predefined time points was        determined.

Virus Neutralization:

Neutralizing activity of induced antibodies was determined by acytopathic effect (CPE)-based micro-neutralization assay looking at 50%CPE by a viral infective dose 25 (MN 25 TCID50/well), using a wild-typeviral strain (SARS-CoV-2 2019 nCOV ITALY/INMI1) on a VERO E6 cell line.The assays were performed in a 96-well plate format, human serum wasdiluted in a 1:2 serial dilution. The Micro-neutralization titre is thereciprocal of the highest sample dilution that protects from CPE atleast 50% of cells and reported as the geometric mean of duplicates.

Elisa:

Antibody titres were measured with Elisa Assays using as target antigeneither the extra cellular domain (ECD) of Spike or to the receptorbinding domain (RBD). The antigen recombinant proteins used for coatingwere expressed in eukaryotic cells. Human serum were diluted 1:2 in aserial dilution, the titre is the reciprocal of the highest sampledilution over a cut-point defined as blank plus matrix effect. Titresare reported as geometric mean of duplicates.

T Cell Responses:

As an exploratory endpoint of this clinical trial, cell-mediated immuneresponses were evaluated by assessment of frequency and functionality ofSARS-CoV-2 Spike-specific CD4+Th1 and cytotoxic CD8+ T cell responsesafter antigen stimulation. Moreover the proportion of subjects with adetectable increase in SARS-CoV-2 spike-specific T-cell responses aftervaccination were determined.

Functional T cell responses were determined and quantified ex vivo byflow cytometry-based intracellular cytokine staining (ICS) of T cellactivation markers and effector cytokines (CD40L, IFN-gamma, IL-2 andTNF-alpha) after stimulation of SARS-CoV-2 Spike-specific CD4+Th1 andcytotoxic CD8+ T cells with overlapping Spike peptide pools.

Results:

In FIG. 13A systemic adverse events are shown in the different dosecohorts after the first dose and after the second dose.

In FIG. 13B local adverse events are shown in the different dose cohortsafter the first dose and after the second dose.

In FIG. 13C the specific systemic adverse events are shown, such asfatigue, headache, myalgia, chills, arthralgia, fever, nausea anddiarrhea.

In FIG. 13D the specific local adverse events are shown, such as pain,itching, swelling and redness.

In summary the CvnCoV vaccine showed good safety properties andacceptable reactogenicity.

In FIG. 13E induction of Spike protein specific IgG antibodies on day 1,29, 36, 43 and 57 is shown for the different dose cohorts. Allvaccinated subjects showed good induction of Spike-specific antibodies,wherein the 12 μg cohort showed the same level of antibodies asseroconverted patients (HCS). In the table of FIG. 13E percentage ofseroconversion of the vaccinated subjects is shown. In most of the casesmore than 90% of the vaccinated subjects showed a more than 2 foldincrease in Spike protein-specific antibodies compared to baseline onday 43. In all dose groups at least 70% of the vaccinated subjectsshowed a more than 4 fold increase in Spike protein-specific antibodiescompared to baseline. In the 12 μg even more than 90% of the subjectsshowed a more than 4 fold increase in antibodies.

In FIG. 13F induction of RBD-specific IgG antibodies on day 1, 36, and43 is shown for the different dose cohorts. All vaccinated subjectsshowed good induction of RBD-specific antibodies, wherein the 12 μgcohort showed the same level of antibodies as seroconverted patients(HCS). In the table of FIG. 13F percentage of seroconversion of thevaccinated subjects is shown. In most of the cases more than 80% of thevaccinated subjects showed a more than 2 fold increase in RBD-specificantibodies compared to baseline on day 43. In the 8 μg and the 12 μggroups more than 80% of the subjects showed a more than 4 fold increasein antibodies.

In FIG. 13G induction of virus neutralizing antibodies is shown. Alldose groups showed good induction of virus neutralizing titers whereinthe highest dose of 12 μg induced the same level of neutralizingantibodies as present in seroconverted patients (HCS). In the table ofFIG. 13G percentage of seroconversion of the vaccinated subjects isshown. In all dose groups more than 70% of the vaccinated subjectsshowed a more than 2 fold increase in virus neutralizing antibodiescompared to baseline on day 43. In the 8 μg and 12 μg dose groups atleast 70% of the vaccinated subjects showed a more than 4 fold increasein virus neutralizing antibodies compared to baseline. In the 12 μg even100% of the subjects showed a more than 4 fold increase in virusneutralizing antibodies.

In FIG. 13H the ratios of the level of Spike protein or RBD bindingantibodies to the level of neutralizing antibodies are shown.Importantly, the CVnCoV induced ratio is about the same as fromconvalescent subjects, which implies that the induced level ofantibodies is sufficient to neutralize SARS-CoV-2.

FIG. 13I shows induction of CD4+ T cells against Spike protein S1 afterthe first dose (day 29) and the second dose (day 36). Both dose groups(4 μg and 8 μg) show good induction of CD4+ T cells against Spikeprotein S1.

FIG. 13J shows induction of CD4+ T cells against Spike protein S2 afterthe first dose (day 29) and the second dose (day 36). Both dose groups(4 μg and 8 μg) show good induction of CD4+ T cells against Spikeprotein S comparable to convalescent patients.

In FIG. 13K induction of virus neutralizing titers in SARS-CoV-2seropositive subjects (upper part) after vaccination with 2 μg (left)and 4 μg (right) CvnCoV is shown. Remarkably, virus neutralizingantibodies could be boosted in both dose groups in seropositive patientsalready expressing virus neutralizing antibodies.

In the lower part induction of RBD specific antibodies in SARS-CoV-2seropositive subjects after vaccination with 2 μg (left) and 4 μg(right) CvnCoV is shown. Remarkably, RBD specific antibodies could beboosted in both dose groups in seropositive patients already expressingRBD specific antibodies.

Example 11: Vaccination of Mice with mRNA Encoding SARS-CoV-2 AntigenS_Stab Formulated in LNPs

The present example shows that SARS-CoV-2 S mRNA vaccines with mRNAcomprising alternative forms of the 3′ end (A64-N5-C30-hSL-N5 orhSL-A100) and UTR combinations (i-3 (−/muag) or a-1 (HSD17B4/PSMB3))induce strong humoral as well as cellular immune response in mice. mRNAencoding SARS-CoV-2 S_stab comprising hSL-A100 and the UTR combinationa-1 (HSD17B4/PSMB3) shows stronger induction of immune responses,demonstrated by a stronger induction of binding and neutralizingantibodies as well as by a stronger induction of CD8+ T-cells.

Preparation of LNP Formulated mRNA Vaccine:

SARS-CoV-2 S mRNA constructs are prepared as described in Example 1 (RNAin vitro transcription). HPLC purified mRNA was formulated with LNPsaccording to Example 1.4.

Immunization:

Female BALB/c mice (6-8 weeks old, n=8) were injected intramuscularly(i.m.) with mRNA vaccine compositions at dosages indicated in Table 16.As a negative control, one group of mice was vaccinated with buffer. Allanimals were vaccinated on day 0 and 21. Blood samples were collected onday 21 (post prime) and 42 (post boost) for the determination ofantibody titers, splenocytes were isolated on day 42 for T-cellanalysis.

TABLE 16 Vaccination regimen (Example 11): 5′-UTR/ 3′-UTR; SEQ SEQVaccine mRNA CDS UTR ID NO: ID NO: Group composition ID opt. Design3′-end Protein RNA Dose A buffer — — — — — B mRNA encoding R9515 opt1—/muag; A64-N5- 10 163 1 μg S_stab formulated C30-hSL- in LNPs N5 C mRNAencoding R9709 opt1 HSD17B4/ hSL- 10 149 1 μg S_stab formulated PSMB3A100 in LNPs

Determination of IgG1 and IgG2 antibody titers using ELISA,determination of virus neutralizing titers via CPE (cytopathic effect)and T-cell analysis by Intracellular cytokine staining (ICS) wasperformed as described in Example 6.

Results:

As shown in FIG. 14A the vaccination with mRNA encoding full length Sstabilized protein (S_stab) induced high titers of S specific bindingantibody (IgG1 and IgG2a) after a single vaccination (d21). The titersincreased after a second vaccination (d42). Vaccine compositioncomprising mRNA encoding SARS-CoV-2 S_stab comprising hSL-A100 and theUTR combination a-1 (HSD17B4/PSMB3) (group C) showed an improved andstronger induction of binding antibodies (shown by IgG1 and IgG2aendpoint titers).

Both mRNA designs induced more or less comparable virus neutralizationantibody titers after second vaccination (day 42), whereas mice of groupC showed an early increased level of VNTs already on d21 after firstvaccination compared to group B (shown in FIG. 14B).

As shown in FIG. 14C the vaccination with mRNA encoding full length Sstabilized protein with both alternative forms of the 3′ end(A64-N5-C30-hSL-N5 or hSL-A100) and UTR combinations (i-3 (−/muag) ora-1 (HSD17B4/PSMB3)) induced robust levels of antigen-specific CD4+ andCD8⁺ IFNγ/TNF double positive T cells after two vaccinations. Vaccinecomposition comprising mRNA encoding SARS-CoV-2 S_stab comprisinghSL-A100 and the UTR combination a-1 (HSD17B4/PSMB3) (group C) showedsurprisingly a remarkable stronger induction of CD8⁺ IFNγ/TNF doublepositive T cells.

Example 12: Vaccination of Rats with mRNA Encoding SARS-CoV-2 AntigenS_Stab Formulated in LNPs

The present example shows that SARS-CoV-2 S mRNA vaccines with mRNAcomprising the inventive alternative form of the 3′ end (hSL-A100) andUTR combination (a-1 (HSD17B4/PSMB3)) induce strong humoral immuneresponse in rats.

Preparation of LNP Formulated mRNA Vaccine:

SARS-CoV-2 S mRNA constructs are prepared as described in Example 1 (RNAin vitro transcription). HPLC purified mRNA was formulated with LNPsaccording to Example 1.4 prior to use in in vivo vaccinationexperiments.

Immunization:

Rats were injected intramuscularly (i.m.) with mRNA vaccine compositionsand doses as indicated in Table 17. As a negative control, one group ofrats was vaccinated with buffer (group A). All animals were vaccinatedon day 0 and day 21. Blood samples were collected on day 21 (post prime)and 42 (post boost) for the determination of antibody titers.

TABLE 17 Vaccination regimen (Example 12): 5′-UTR/ 3′-UTR; SEQ SEQ mRNACDS UTR ID NO: ID NO: Group Vaccine composition ID opt. Design 3′-endProtein RNA Dose A buffer — — — — — B mRNA encoding S_stab R9709 opt1HSD17B4/ hSL- 10 149 0.5 μg formulated in LNPs PSMB3 A100 C mRNAencoding S_stab R9709 opt1 HSD17B4/ hSL- 10 149   2 μg formulated inLNPs PSMB3 A100 C mRNA encoding S_stab R9709 opt1 HSD17B4/ hSL- 10 149  8 μg formulated in LNPs PSMB3 A100

Determination of IgG1 and IgG2 Antibody Titers Using ELISA:

ELISA was performed using recombinant SARS-CoV-2 S (receptor bindingdomains RBD) protein for coating. Coated plates were incubated usingrespective serum dilutions, and binding of specific antibodies toSARS-CoV-2 S were detected directly with labeled HRP antibody instead ofa secondary HRP antibody used for mouse ELISA. The lack of signalamplification in rat ELISA might account for lower titers, thereforeELISA titers between rat and mouse studies are currently not comparable.

Determination of Virus Neutralizing Antibody Titers (VNT)

Virus neutralizing antibody titers (VNT) of rat serum samples wereanalyzed as previously described in Example 6 with mouse serum.

Results:

As shown in FIG. 15A the vaccination with mRNA full length S stabilizedprotein comprising the alternative non-coding region with 3′ endhSL-A100 and the UTR combination a-1 (HSD17B4/PSMB3) formulated in LNPsinduced in rats robust and dose dependent levels of binding antibodytiters at day 21 after first vaccination and at day 42 after secondvaccination using doses of 0.5 μg, 2 μg and 8 μg. The second vaccinationled to a further increase of antibody titers.

As shown in FIG. 15B vaccination with mRNA comprising the alternativeand inventive non-coding region with 3′ end hSL-A100 and the UTRcombination a-1 (HSD17B4/PSMB3) encoding full length S stabilizedprotein and formulated in LNPs induced in rats dose dependent and veryhigh levels of VNT. The humoral immune responses shown by ELISA (bindingantibodies IgG1 and IgG2a) and VNTS are remarkably increased compared tothe immune responses elicited with mRNA comprising non-coding regionwith 3′ end A64-N5-C30-hSL-N5 and UTR combination i-3 (−/muag) (see forcomparison Example 8 FIG. 11A-F).

Example 13: Clinical Development of SARS-CoV-2 (CVnCoV) Vaccine

1 Trial Protocol for Human Vaccination

2 Summary

The trial is designed as a Phase 2b/3 pivotal efficacy and safety trialin adults 18 years of age and older. The trial will have a randomized,observer-blinded, placebo-controlled design. Subjects will be enrolledat multiple sites globally and will be randomized in a 1:1 ratio toreceive a 2-dose schedule of either CVnCoV at a dose level of 12 μg mRNAor placebo {normal saline (0.9% NaCl)} as the control.

3 Extension

Following completion of Trial CV-NCOV-004 on Day 393, subjects willcontinue to participate in a 1 year extension of the trial. At the timeof consent for Trial CV-NCOV-004, subjects will also be consented forparticipation in the 1 year extension. The Extension Study will collectadditional data to evaluate long term safety {serious adverse events(SAEs)}, persistence of antibodies to SARS-CoV-2, and the occurrence ofCOVID-19 cases to assess duration of vaccine efficacy (VE).

4 Trial Objectives, Endpoints, and Estimands

4.1 Objectives

4.1.1 Primary Objectives

Co-Primary Efficacy Objectives

-   -   To demonstrate the efficacy of a 2-dose schedule of CVnCoV in        the prevention of first episodes of virologically-confirmed        cases of COVID-19 of any severity in SARS CoV 2 naïve subjects.    -   To demonstrate the efficacy of a 2-dose schedule of CVnCoV in        the prevention of first episodes of virologically-confirmed        moderate to severe cases of COVID-19 in SARS CoV-2 naïve        subjects.

Primary Safety Objective

-   -   To evaluate the safety of CVnCoV administered as a 2-dose        schedule to subjects 18 years of age and older.

4.1.2 Secondary Objectives

Key Secondary Efficacy Objectives

-   -   To demonstrate the efficacy of a 2-dose schedule of CVnCoV in        the prevention of first episodes of virologically-confirmed        severe cases of COVID-19 in SARS-CoV-2 naïve subjects.    -   To demonstrate the efficacy of a 2-dose schedule of CVnCoV in        the prevention or reduction of asymptomatic infection by        SARS-CoV-2 in seronegative subjects, as measured by        seroconversion to the N protein of the virus.

Other Secondary Efficacy Objectives

To evaluate in SARS-CoV-2 naïve subjects:

-   -   The efficacy of a 2-dose schedule of CVnCoV in the prevention of        first episodes of virologically-confirmed cases of COVID-19 of        any severity in subjects≥61 years of age.    -   The efficacy of a 2-dose schedule of CVnCoV in the prevention of        first episodes of virologically-confirmed cases of SARS-CoV-2        infection, with or without symptoms.    -   The efficacy of a 2-dose schedule of CVnCoV in reducing the        Burden of disease (BoD) from COVID-19.    -   The efficacy of CVnCoV after the first dose in the prevention of        first episodes of virologically-confirmed cases of COVID-19 of        any severity.

Secondary Immunogenicity Objectives

-   -   To assess antibody responses to the RBD of S protein of        SARS-CoV-2 after 1 and 2 doses of CVnCoV in a subset of subjects        participating in Phase 2b of the trial.    -   To assess SARS-CoV-2 viral neutralizing antibody responses after        1 and 2 doses of CVnCoV in a subset of subjects participating in        Phase 2b of the trial.

Secondary Safety Objective

-   -   To evaluate the reactogenicity and tolerability of CVnCoV        administered as a 2-dose schedule to subjects 18 years of age        and older participating in Phase 2b of the trial.

4.1.3 Exploratory Objectives

Exploratory Efficacy Objectives

To investigate in SARS-CoV-2 naïve subjects:

-   -   If cases of COVID-19 are milder in severity in subjects        receiving a 2-dose schedule of CVnCoV compared to those        administered placebo.    -   If the need for supplemental oxygenation due to COVID-19 is        reduced in subjects receiving a 2-dose schedule of CVnCoV        compared to those administered placebo.    -   If the need for mechanical ventilation due to COVID-19 is        reduced in subjects receiving a 2-dose schedule of CVnCoV        compared to those administered placebo.    -   If hospitalization due to COVID-19 is reduced in subjects        receiving a 2-dose schedule of CVnCoV compared to those        administered placebo.    -   If mortality due to COVID-19 is reduced in subjects receiving a        2-dose schedule of CVnCoV compared to those administered        placebo.    -   If all-cause mortality is reduced in subjects receiving a 2-dose        schedule of CVnCoV compared to those administered placebo.    -   To investigate the cell-mediated immune (CMI) response of a        2-dose schedule of CVnCoV from up to 400 subjects at selected        site(s).

To investigate in SARS-CoV-2 naïve and non-naïve subjects:

-   -   The efficacy of a 2-dose schedule of CVnCoV in the prevention of        first episodes of virologically-confirmed cases of COVID-19 of        any severity in all subjects, regardless of SARS-CoV-2        serological status at baseline.    -   The efficacy of CVnCoV after the first dose in the prevention of        first episodes of virologically-confirmed cases of COVID-19 of        any severity in all subjects, regardless of SARS-CoV-2        serological status at baseline.

To investigate in subjects with first episodes ofvirologically-confirmed COVID-19 during the trial:

-   -   The occurrence of second episodes of COVID-19 in subjects        receiving a 2-dose schedule of CVnCoV compared to those        administered placebo.

4.2 Endpoints

4.2.1 Primary Endpoints

Co-Primary Efficacy Endpoints

-   -   Occurrence of first episodes of virologically-confirmed {reverse        transcription polymerase chain reaction (RT-PCR) positive} cases        of COVID-19 of any severity meeting the case definition for the        primary efficacy analysis.    -   Occurrence of first episodes of virologically-confirmed (RT-PCR        positive) cases of moderate to severe COVID-19 meeting the case        definition for the primary efficacy analysis (moderate and        severe COVID-19 disease defined herein).

Primary Safety Endpoints

-   -   Occurrence, intensity and relationship of medically-attended AEs        collected through 6 months after the second trial vaccination in        all subjects.    -   Occurrence, intensity and relationship of SAEs and AESIs        collected through 1 year after the second trial vaccination in        all subjects.    -   Occurrence of fatal SAEs through 1 year after the second trial        vaccination in all subjects.

4.2.2 Secondary Endpoints

Key Secondary Efficacy Endpoints

-   -   Occurrence of first episodes of virologically-confirmed (RT-PCR        positive) severe cases of COVID-19 meeting the case definition        for the primary efficacy analysis (severe COVID-19 disease        defined in herein).    -   Occurrence of seroconversion to the N protein of SARS-CoV-2≥15        days following the second trial vaccination in asymptomatic        seronegative subjects.

Seroconversion is defined as detectable SARS-CoV-2 N protein antibodiesin the serum of subjects on Day 211 and/or Day 393 of the trial, whotested seronegative at Day 1 (baseline) and Day 43 (i.e. at the 2testing time points prior to 15 days following the second trialvaccination).

Other Secondary Efficacy Endpoints

-   -   In subjects≥61 years of age, occurrence of first episodes of        virologically-confirmed (RT-PCR positive) cases of COVID-19 of        any severity meeting the case definition for the primary        efficacy analysis.    -   Occurrence of virologically-confirmed (RT-PCR positive)        SARS-CoV-2 infection, with or without symptoms.

If subject was symptomatic, onset of symptoms must have occurred 15 daysfollowing the second trial vaccination; if subject was asymptomatic, thepositive RT PCR test must have occurred 15 days following the secondtrial vaccination.

-   -   BoD scores calculated based on first episodes of        virologically-confirmed (RT-PCR positive) cases of COVID-19 of        any severity meeting the case definition for the primary        efficacy analysis.        -   BoD #1—no disease (not infected or asymptomatic            infection)=0; mild or moderate disease=1; severe disease=2.        -   BoD #2—no disease (not infected or asymptomatic            infection)=0; disease without hospitalization=1; disease            with hospitalization=2; death=3.    -   Occurrence of first episodes of virologically-confirmed (RT-PCR        positive) cases of COVID-19 of any severity with symptom onset        at any time after the first trial vaccination.

Secondary Immunogenicity Endpoints (Phase 2b Immunogenicity Subset)

SARS-CoV-2 RBD of S Protein Antibody Responses

On Days 1, 29, 43, 57, 120, 211 and 393:

-   -   Serum antibodies to SARS-CoV-2 RBD of S protein.    -   Occurrence of seroconversion to SARS-CoV-2 RBD of S protein.

Seroconversion is defined as detectable SARS-CoV-2 RBD of S proteinantibodies in the serum of subjects who tested seronegative at baseline.

SARS-CoV-2 Viral Neutralizing Antibody Responses

On Days 1, 29, 43, 57, 120, 211, and 393:

-   -   Serum viral neutralizing antibodies to SARS-CoV-2 virus, as        measured by a viral neutralizing antibody assay.    -   Occurrence of seroconversion to SARS-CoV-2 virus, as measured by        a viral neutralizing antibody assay. Seroconversion is defined        as detectable SARS-CoV-2 viral neutralizing antibodies in the        serum of subjects who tested seronegative at baseline.

Secondary Safety Endpoints

-   -   Occurrence, intensity and duration of each solicited local AE        within 7 days after each trial vaccination in Phase 2b subjects.    -   Occurrence, intensity, duration of each solicited systemic AE        within 7 days after each trial vaccination in Phase 2b subjects.    -   Occurrence, intensity and relationship of unsolicited AEs        occurring within 28 days after each trial vaccination in Phase        2b subjects.    -   Occurrence of AEs leading to vaccine withdrawal or trial        discontinuation through 1 year after the second trial        vaccination in all subjects.

4.2.3 Exploratory Endpoints

Exploratory Efficacy Endpoints

-   -   Severity assessment of first episodes of virologically-confirmed        (RT-PCR positive) cases of COVID-19 meeting the case definition        for the primary efficacy analysis.

The following endpoints will be analyzed as occurring 15 days followingthe second trial vaccination (full VE) and at any time after the firsttrial vaccination.

In SARS-CoV-2 naïve subjects:

-   -   Occurrence of supplemental oxygenation due to COVID-19 disease.    -   Occurrence of mechanical ventilation due to COVID-19 disease.    -   Occurrence of hospitalization due to COVID-19 disease.    -   Occurrence of death due to COVID-19 disease.    -   Occurrence of death due to any cause.

In SARS-CoV-2 naïve and non-naïve subjects:

-   -   In all subjects regardless of their baseline serostatus,        occurrence of first episodes of virologically-confirmed (RT-PCR        positive) cases of COVID-19 of any severity.

The following endpoint will be analyzed in subjects who had a firstepisode of a virologically-confirmed (RT-PCR positive) case of COVID-19of any severity meeting the case definition for the primary efficacyanalysis.

-   -   Occurrence of second episodes of virologically-confirmed (RT-PCR        positive) cases of COVID-19 of any severity.

Exploratory Immunogenicity Endpoints (Phase 2b Immunogenicity Subset)

On Days 1, 29, 43, 120**, and 211** in peripheral blood mononuclearcells (PBMCs) from up to 400 subjects at selected site(s):

-   -   The frequency and functionality of SARS-CoV-2 RBD of S-specific        T-cell response after antigen stimulation by intracellular        cytokine staining (ICS) to investigate Th1 response and        expression of Th2 markers.    -   The proportion of subjects with a detectable increase in        SARS-CoV 2 RBD of S specific T cell response.

**Testing of samples collected on Day 120 and Day 211 will be done onlyin subjects categorized as T-cell responders on Day 29 and/or Day 43.

4.3 Estimands

TABLE 16 ENDPOINTS (subject level) ESTIMANDS (population level)Co-Primary Efficacy Occurrence of first episodes In naïve evaluablesubjects of virologically-confirmed (complying with the definition(RT-PCR positive) cases of of Efficacy Analysis Set) at COVID-19 of anyseverity least 15 days following meeting the case definition for secondvaccination: the primary efficacy analysis. VE = 1-RR with exact 97.5%CI Occurrence of first episodes Where RR (relative risk) is ofvirologically-confirmed the ratio of attack rates of (RT-PCR positive)cases of COVID-19 cases per 100 moderate to severe COVID- person-monthin the CVnCoV 19 meeting the case definition vaccine group over the forthe primary efficacy analysis. placebo group. Primary Safety Occurrence,intensity and In subjects who received at least relationship ofmedically- one dose of CVnCoV attended AEs collected through or placebovaccine, the 6 months after the second number and percentage of trialvaccination in all subjects. subjects by group reporting at Occurrence,intensity and least 1 and at each type relationship of SAEs and (bySOC/PT) of: AESIs collected through 1 Medically-attended AE in the 6year after the second trial months after the last vaccination in allsubjects. vaccination overall, by Occurrence of fatal SAEs intensity andby causal through 1 year after the relationship to trial vaccine. secondtrial vaccination in SAE in the year after the last all subjects.vaccination overall and by causal relationship to trial vaccine. AESI inthe year after the last vaccination overall, by intensity and by causalrelationship to trial vaccine. Fatal SAE in the year after the lastvaccination. Key Secondary Efficacy Occurrence of first episodes Innaïve evaluable subjects of virologically-confirmed (complying with thedefinition (RT-PCR positive) severe of Efficacy Analysis Set) at casesof COVID-19 meeting least 15 days following the case definition for thesecond vaccination: primary efficacy analysis. VE = 1-RR with exact 95%CI Where RR (relative risk) is the ratio of attack rates of severeCOVID-19 cases per 100 person-month in the CVnCoV vaccine group over theplacebo group. Occurrence of seroconversion In naïve evaluable subjectsto the N protein of SARS- (complying with the definition CoV-2 ≥15 daysfollowing of Efficacy Analysis Set) who the second trial vaccinationtested seronegative at in asymptomatic seronegative baseline and Day 43for the subjects. N protein of SARS-COV-2  Seroconversion is defined andwith at least 1 of Day 211  as detectable SARS- or Day 393 serologydone:  CoV-2 N protein antibodies VE = 1-RR with exact 95% CI  in theserum of subjects Where RR (relative risk) is  on Day 211 and/or Day theratio of attack rates of  393 of the trial, who tested Asymptomaticinfections  seronegative at Day 1 (Seroconversion to the N  (baseline)and Day 43 (i.e. protein at Day 211 and then  at the 2 testing timepoints seroconversion to the N  prior to 15 days following protein ateither Day 211 or  the second trial vaccination). Day 393) in the CVnCoVvaccine group over the placebo group. Other Secondary Efficacy Insubjects ≥61 years of In naïve evaluable subjects age, occurrence offirst ≥61 years of age at episodes of virologically- randomization(complying confirmed (RT-PCR positive) with the definition of Efficacycases of COVID-19 of any Analysis Set) at least 15 severity meeting thecase days following second definition for the primary vaccination:efficacy analysis. VE = 1-RR with exact 95% CI Where RR (relative risk)is the ratio of attack rates of COVID-19 cases per 100 person-month inthe CVnCoV vaccine group over the placebo group. Occurrence ofvirologically- In naïve evaluable subjects confirmed (RT-PCR (complyingwith the definition positive) SARS-CoV-2 of Efficacy Analysis Set) atinfection, with or without least 15 days following symptoms. secondtrial vaccination:  If subject was symptomatic, VE = 1-RR with exact 95%CI  onset of symptoms Where RR (relative risk) is  must have occurred≥15 days the ratio of attack rates of  following the secondvirologically-confirmed (RT-PCR  trial vaccination; if subject positive)SARS-CoV-2  was asymptomatic, the infection per 100 person-month positive RT-PCR test in the CVnCoV vaccine  must have occurred ≥15group over the placebo group.  days following the second  trialvaccination. BoD scores calculated In naïve evaluable subjects based onfirst episodes of (complying with the definition virologically-confirmedof Efficacy Analysis Set) at (RT-PCR positive) cases of least 15 daysfollowing COVID-19 of any severity second trial vaccination: meeting thecase definition VE = 1-RR with exact 95% CI for the primary efficacyanalysis. Where RR (relative risk) is   BoD #1-no disease the ratio ofattack rates of   (not infected or virologically-confirmed (RT-  asymptomatic infection) = 0; PCR positive) SARS-CoV-2   mild ormoderate infection per 100 person-month   disease = 1; severe disease =2. in the CVnCoV vaccine   BoD #2-no disease group over the placebogroup   (not infected or   asymptomatic infection) = 0;   diseasewithout   hospitalization = 1;   disease with   hospitalization = 2;death = 3. Occurrence of first episodes In naïve subjects who receivedof virologically-confirmed at least one dose of (RT-PCR positive) casesof CVnCoV or placebo vaccine COVID-19 of any severity at any time afterthe first with symptom onset at any vaccination: time after the firsttrial VE = 1-RR with exact 95% CI vaccination. Where RR (relative risk)is the ratio of attack rates of COVID-19 cases per 100 person-month inthe CVnCoV vaccine group over the placebo group Secondary ImmunogenicitySARS-CoV-2 RBD of S protein In phase 2b subjects belonging antibodyresponses to the Immunogenicity On Days 1, 29, 43, 57, 120, subset andevaluable 211 and 393: (complying with the definition of  Serumantibodies to SARS- per-protocol immunogenicity set):  CoV-2 RBD ofspike (S) On Days 1, 29, 43, 57,  protein, as measured by 120, 211 and393:  enzyme-linked immunosorbent  Geometric mean of titers  assay(ELISA).  (GMT) with 95% CI of SARS-CoV-2 RBD of S  SARS-CoV-2 RBD ofspike protein antibody responses  (S) protein antibody On Days 1, 29,43, 57,  responses by group and by 120, 211 and 393:  baselinesero-status  Occurrence of seroconversion  and group   to SARS-CoV-2 RBDof On Days 29, 43, 57, 120, 211  spike (S) protein, as and 393 forsubjects  measured by ELISA. seropositive at baseline:   Seroconversionis defined  Geometric mean of Fold   as detectable SARS-  Change frombaseline   CoV-2 RBD of spike (S)  (GMFC) with 95% CI of   proteinantibodies in the  SARS-CoV-2 RBD of spike   serum of subjects who  (S)protein antibody   tested seronegative at  responses by group.  baseline. On Days 29, 43, 57, 120, 211 and 393 for subjectsseronegative at baseline:  Number and percentage  with exact 95% CI of subjects by group for who  a seroconversion is  observed (detectable SARS-CoV-2 RBD of S  protein antibodies in the serum). SARS-CoV-2 viralneutralizing In phase 2b subjects belonging antibody responses (subsetto the Immunogenicity of subjects analyzed) subset and evaluable(complying On Days 1, 29, 43, 57, with the definition of 120, 211 and393: per-protocol immunogenicity set):  Serum viral neutralizing On Days1, 29, 43, 57, 120,  antibodies to SARS-CoV-2 211 and 393:  virus, asmeasured by a  Geometric mean of titers  viral neutralizing antibody (GMT) with 95% CI of  assay.  neutralizing antibodies SARS-CoV-2 viralneutralizing  to SARS-CoV-2 virus by antibody responses (subset  groupand by baseline of subjects analyzed)  serostatus and group On Days 1,29, 43, 57, 120, 211 and 393: Occurrence of seroconversion On Days 29,43, 57, 120, 211 to SARS-CoV-2 virus, as and 393 for subjects measuredby a viral seropositive at baseline: neutralizing antibody assay. Geometric mean of Fold  Seroconversion is defined  Change from baseline as detectable SARS-  (GMFC) with 95% CI of  CoV-2 viral neutralizing neutralizing antibodies to  antibodies in the serum of  SARS-CoV-2virus by group.  subjects who tested On Days 29, 43, 57, 120, seronegative at baseline. 211 and 393 for subjects seronegative atbaseline:  Number and percentage  with exact 95% CI of  subjects bygroup for who  a seroconversion is  observed (detectable  neutralizingantibodies to  SARS-CoV-2 virus in  the serum). Secondary SafetyOccurrence, intensity and In phase 2b subjects who received duration ofeach solicited at least one dose of local AE within 7 days after CVnCoVor placebo vaccine: each trial vaccination in The number and percentagePhase 2b subjects. of subjects by group Occurrence, intensity,reporting: duration of each solicited  Each solicited local AE withinsystemic AE within 7 days  7 days (after each trial after each trialvaccination in  vaccination by intensity Phase 2b subjects.  and overallOccurrence, intensity and  Each solicited systemic AE relationship ofunsolicited  within 7 days after each AEs occurring within 28 days trial vaccination by intensity, after each trial vaccination  byrelationship to trial in Phase 2b subjects.  vaccine and overall.Occurrence of AEs leading to  At least 1 unsolicited AEs, vaccinewithdrawal or trial  at least 1 grade 3 unsolicited discontinuationthrough 1  AEs and each unsolicited year after the second trial  AEs bySOC/PT occurring vaccination in all subjects.  within 28 days after eachtrial  vaccination and overall by  causal relationship to trial  vaccineand overall  At least 1 AEs leading to  vaccine withdrawal or trial discontinuation in the  year after the last trial  vaccination The meanduration in days by group with standard deviation of solicited AEs(within the solicited period, total duration). Exploratory EfficacySeverity assessment of first In naïve evaluable subjects episodes ofvirologically- (complying with the definition confirmed (RT-PCRpositive) of Efficacy Analysis Set) cases of COVID-19 who had a firstepisode of a meeting the case definition virologically-confirmed for theprimary efficacy (RT-PCR positive) case of analysis COVID-19 of anyseverity meeting the case definition for the primary efficacy analysis: The proportions of mild and  severe COVID-19 cases  among all cases bygroup The following endpoints will In naïve evaluable subjects beanalyzed as occurring ≥15 (complying with the definition days followingthe second trial of Efficacy Analysis Set) vaccination (full vaccine atleast 15 days following efficacy) and at any time after secondvaccination AND the first trial vaccination. In subjects who received at Occurrence of supplemental least one dose of CVnCoV  oxygenation due toor placebo vaccine at any  COVID-19 disease. time after the first trial Occurrence of mechanical vaccination:  ventilation due to COVID-19Number and percentages  disease. by group of subjects who:  Occurrenceof hospitalization  Need for supplemental  due to COVID-19  oxygenationdue to COVID-19.  disease.  Need for mechanical  Occurrence of death due ventilation due to COVID-19.  to COVID-19 disease.  Hospitalized due toCOVID-19.  Occurrence of death  Deceased due to COVID-19.  due to anycause.  Deceased due to any cause.  In all subjects regardless Insubjects who received at  of their baseline serostatus, least one doseof CVnCoV  occurrence of first episodes or placebo vaccine, at any  ofvirologically-confirmed time after the first trial  (RT-PCR positive)cases of vaccination:  COVID-19 of any severity. VE = 1-RR with exact95% CI Where RR (relative risk) is the ratio of attack rates of COVID-19cases per 100 person-month in the CVnCoV vaccine group over the placebogroup The following endpoint will In naïve evaluable subjects beanalyzed in subjects who had (complying with the definition a firstepisode of a virologically- of Efficacy Analysis Set) who confirmed(RT-PCR positive) had a first episode of a case of COVID-19 of anyvirologically-confirmed severity meeting the case (RT-PCR positive) caseof definition for the primary COVID-19 of any severity efficacyanalysis. meeting the case definition for  Occurrence of second theprimary efficacy analysis,  episodes of virologically- at least 15 daysfollowing  confirmed (RT-PCR positive) second vaccination:  cases ofCOVID-19 of any  The number and percentage  severity.  of subjects who developed a second  episode of COVID-19. Exploratory Immunogenicity OnDays 1, 29, 43, 120**, and Exploratory immunogenicity 211** inperipheral blood estimands will be described mononuclear cells (PBMCs)in the Statistical Analysis from up to 400 subjects at Plan, asapplicable. selected site(s):  The frequency and functionality  ofSARS-CoV-2 RBD of  S-specific T-cell response  after antigen stimulationby  intracellular cytokine staining  (ICS) to investigate Th1  responseand expression  of Th2 markers.  The proportion of subjects  with adetectable increase in  SARS-CoV-2 RBD of  S-specific T-cell response.** Testing of samples collected on Day 120 and Day 211 will be done onlyin subjects categorized as T-cell responders on Day 29 and/or Day 43.

5 Trial Design

5.1 Overall Design

Trial CV-NCOV-004 will be conducted in 2 parts: an initial Phase 2btrial followed by transition to a large Phase 3 efficacy trial. BothPhase 2b and Phase 3 will be conducted as randomized, observer-blinded,placebo-controlled trials. Subjects 18 years of age or older will beenrolled at multiple sites globally and will receive a 2-dose scheduleof either CVnCoV at a dose level of 12 μg mRNA or placebo {normal saline(0.9% NaCl)} in a 1:1 ratio. Both Phase 2b and Phase 3 parts of thetrial are consistent in design (e.g., for COVID-19 case ascertainmentand case definition) so that cases of COVID-19 occurring in Phase 2b canbe pooled with those in Phase 3 for the primary analysis of VE. Subjectswill also participate in a 1-year extension of the Phase 2b and Phase 3parts of the trial.

Phase 2b Design and Objectives

The objective of Phase 2b is to further characterize the safety,reactogenicity, and immunogenicity of CVnCoV prior to initiating Phase3. CVnCoV will be administered at the 12 μg dose level selected forPhase 3 investigation informed by the safety and immunogenicity datafrom the initial Phase 1 and 2a trials. Phase 2b will be conducted in 2age groups of adults: 18 to 60 and ≥61 years of age, which represent theage range of the intended Phase 3 trial population.

Approximately 4,000 subjects will be enrolled and randomized in a 1:1ratio to receive 2 doses of either CVnCoV at a dose level of 12 μg mRNAor placebo, administered 28 days apart. Of the 4,000 subjects enrolled,approximately 800 to 1,000 (20% to 25%) will be 61 years of age. Phase2b will be performed in an observer-blinded manner to reduce anypotential bias in the safety assessments. The sample size of 4,000subjects is based on generating a robust and detailed datasetcharacterizing the safety, reactogenicity, and immunogenicity of CVnCoVprior to entering Phase 3. Furthermore, the data generated in Phase 2bwill be the main dataset to be submitted in support of early conditionalapproval of CVnCoV.

In Phase 2b, the safety and reactogenicity of a 2-dose schedule ofCVnCoV will be assessed in detail by measuring the frequency andseverity of the following AEs: solicited local and systemic reactionsfor 7 days after each vaccination; unsolicited AEs for 28 days aftereach vaccination; medically-attended AEs through 6 months after thesecond trial vaccination; and AESIs and SAEs through 1 year after thesecond trial vaccination. The immunogenicity of CVnCoV will be evaluatedafter 1 and 2 doses in a subset of subjects (first 600 subjects enrolledin each of the 2 age groups; a total of 1,200 subjects in theImmunogenicity Subset) by measuring binding antibodies to the SARS-CoV-2RBD of S protein and viral neutralizing antibodies. Antibody persistencewill be evaluated in this trial as well as in the Extension Study.

Cases of COVID-19 occurring in Phase 2b subjects will be collected andpooled with those occurring in Phase 3 and the total number of caseswill be used for the primary analysis of efficacy. In addition, the DSMBwill periodically monitor COVID-19 cases for signals of VDE.

Subjects participating in Phase 2b will also be evaluated forasymptomatic SARS-CoV-2 infection during the trial, as measured by thedevelopment of antibodies to the N protein of SARS-CoV-2 (i.e.seroconversion). These data will be combined with those from Phase 3 todetermine if vaccination with CVnCoV can prevent or reduce the rate ofasymptomatic infection by SARS-CoV-2 (one of the key secondary efficacyobjectives).

Initiation of subject enrollment of the 2 target age groups into Phase2b will be flexible. Depending on the timing of data from the Phase 1and Phase 2a trials, enrollment of the 2 age groups into Phase 2b may bestaggered, initially starting with subjects 18 to 60 years of agefollowed by subjects≥61 years of age. As the older age group willcomprise 20% to 25% of the total number of subjects in Phase 2b, thisstaggered start is not expected to impact overall enrollment of thePhase 2b cohort.

An early safety review of the Phase 2b data will be performed by theDSMB (see Section 9.3.9.1). The safety review will be conducted whenapproximately 1,000 subjects have been enrolled in Phase 2b (25% ofsubjects enrolled; 500 recipients of CVnCoV and 500 recipients ofplacebo) and have at least 1 week of safety follow-up after the firsttrial vaccination. If the safety profile is judged to be acceptable andthere are no safety or tolerability concerns, it is anticipated thatenrollment of subjects into Phase 3 can begin without interruption fromPhase 2b. Another safety review by the DSMB will be conducted whenapproximately 1,000 Phase 2b subjects have received their second trialvaccination and have at least week of safety follow-up. All availablefirst dose safety data from the Phase 2b subjects will also be reviewedat this time.

Phase 3 Design and Objectives

The co-primary objectives of the combined Phase 2b/3 trial are todemonstrate the efficacy of a 2-dose schedule of CVnCoV in theprevention of COVID-19 cases of any severity or COVID-19 cases ofmoderate or higher severity. Similar to Phase 2b, Phase 3 will beconducted as a randomized, observer-blinded, placebo controlled trial.Approximately 32,500 subjects, 18 years of age or older, will beenrolled at multiple sites globally in Phase 3 and will receive a 2-doseschedule of either CVnCoV at the 12 μg dose level or placebo in a 1:1ratio (see FIG. 2). Similar to Phase 2b, enrollment will targetsubjects≥61 years of age to be approximately 20% to 25% of the Phase 3trial population (6,500 to 8,125 subjects). The total enrollment of thecombined Phase 2b and Phase 3 parts of the trial will be 36,500subjects.

Subjects will undergo active surveillance for COVID-19. During all sitevisits and phone calls, subjects will be reminded to contact the site ifthey have an acute illness with any symptoms clinically consistent withCOVID-19. In addition, subjects will be messaged up to twice a week andwill provide a yes or no response to having COVID-19 symptoms. Those whorespond “yes” will be contacted by trial staff for a follow-up interviewand assessment. If a subject is suspected of having COVID-19 illness,he/she will undergo testing for SARS-CoV-2 infection with samplescollected at the site or at a home visit. If the subject is confirmed tohave COVID-19, all subjects will be followed until resolution of theirdisease. If the subject is hospitalized, the subject's progress mustcontinue to be followed by the Investigator and a discharge summaryobtained at the end of the hospitalization. Information on clinicalsymptoms and signs, their duration and severity, and treatment andoutcome of the COVID-19 episode will be documented by trial staff andrecorded in the electronic case report form (eCRF). Upon resolution,subjects will continue to be followed through the trial end in the samemanner as those who have not presented with COVID-19. A second episodeof COVID-19 in a subject with prior disease will not be counted as aprimary efficacy case, but will be counted for the exploratory objectiveassessing the reoccurrence of COVID-19 in vaccinated subjects.

Due to the uncertain incidence rate of COVID-19 cases in a pandemicsetting, the trial will be conducted as a case-driven trial based on theany severity COVID-19 endpoint, which will include a two interimanalyses and a final analysis both triggered by achieving a predefinednumber of cases for each analysis. As described above, cases of COVID-19occurring in Phase 2b will be pooled with those in Phase 3 for theprimary analysis of VE. As such, subjects participating in Phase 2b willcontribute to the total sample size for the primary analysis of VE(N=36,500). For the primary analysis of efficacy, the case must meet thefollowing criteria (moderate and severe COVID-19 disease is definedherein):

-   -   Must be a virologically-confirmed case of COVID-19 defined as a        positive SARS CoV 2 specific RT-PCR test in a person with        clinically symptomatic COVID-19 (see Section 9.2).    -   Symptom onset must have occurred 15 days following the second        trial vaccination.    -   The subject must not have a history of virologically-confirmed        COVID-19 at enrollment (based on exclusion criterion

1) or have developed a case of virologically-confirmed COVID-19 before15 days following the second trial vaccination.

-   -   The subject must have been demonstrated to be SARS-CoV-2 naïve        at baseline and at Day 43 (seronegative to N protein).

Primary efficacy cases must be confirmed by the Adjudication Committee.

This trial will utilize a group sequential design with 2 interimanalyses for high efficacy or futility using the O'Brien and Flemingerror spending function for the co-primary endpoint ofvirologically-confirmed COVID-19 cases of any severity. With an overall2-sided alpha of 2.5% and a total of 185 COVID-19 cases of any severitymeeting the primary efficacy case definition at the final analysis, thetrial will have an overall power of 90% to demonstrate a VE greater than30% {based on a margin of 30% for the lower bound of the 97.5%confidence interval (CI) for VE} when considering VE is 60%. Two interimanalyses of high efficacy or futility will be performed once 56/111cases meeting the primary case definition have been accrued (30%/60% offinal case number). These points were chosen based on 2 criteria: i) therobustness of 56/111 cases to support the decision of high efficacy orfutility and ii) if high efficacy, this would shorten the duration ofthe trial and potentially allow the vaccine to be available earlier.

For the co-primary endpoint of virologically-confirmed moderate tosevere COVID-19 cases, with an overall 2-sided alpha of 2.5% and a totalof 60 severe to moderate COVID 19 cases meeting the primary efficacycase definition at the final analysis, the trial will have an overallpower of 90% to demonstrate a VE greater than 20% {based on a margin of20% for the lower bound of the 97.5% confidence interval (CI) for VE}when considering VE is 70%. Assuming that 1/3 of COVID-19 cases aremoderate to severe, 60 moderate to severe cases will be obtained whenthe total number of COVID-19 cases is 180. There will be no interimanalysis for this co-primary endpoint.

Assuming an incidence rate of COVID-19 of 0.15% per month (1.5cases/1000/month) in placebo subjects; a VE of 60%; and a non-evaluablerate of 20% during the trial which includes ˜5% seropositivity ofenrollees at baseline (i.e. non-naïve subjects), follow-up of 36,500subjects enrolled over 3 months (18,250 per vaccine group) will accruethe target 185 COVID-19 cases of any severity approximately 9 monthsafter the first vaccination.

At or near the completion of enrollment, an unblinded review of theincidence rate of cases will be performed by the DSMB. If the caseaccrual rate is lower than expected, the DSMB may recommend an increasein sample size. If needed, another unblinded review by the DSMB may beperformed later in the trial to further adjust the sample size. Thetrial events are shown in the timeline below (the 1-year Extension Studyis discussed below).

With an equal follow-up time of evaluable subjects in both groups,efficacy would be demonstrated at the final analysis if 60 cases or lessof the 185 total cases are in the CVnCoV group (estimated VE 52.0%). Twointerim analyses for high efficacy or futility will be performed when56/111 cases meeting the primary case definition have been accrued(approximately 5/6.5 months after trial start). If the follow-up time ofevaluable subjects is equal in both groups, early high efficacy would bedemonstrated if 7/29 cases or less of the 56/111 cases are in the CVnCoVgroup (estimated VE at interim 85.7%/64.6%); conversely, futility wouldbe reached if 26/41 cases or more are in the CVnCoV group (estimated VEat interim 13.3%/41.4%). The assessment of the interim analyses will beperformed by the DSMB and the outcome will be communicated withoutunblinding the Trial Team or the Sponsor.

Similar to Phase 2b, subjects participating in Phase 3 will be evaluatedfor SARS-CoV-2 infection during the trial, as measured by thedevelopment of antibodies to the N protein of SARS-CoV-2 in seronegativesubjects.

The safety objective of Phase 3 is to generate a large-scale safetydatabase that will demonstrate the safety of CVnCoV. All subjectsparticipating in the Phase 2b and Phase 3 parts of the trial will havemedically-attended AEs collected for 6 months after the secondvaccination; and AESIs and SAEs collected for 1 year after the secondvaccination.

Independent of the demonstration of CVnCoV efficacy at either of theinterim analyses or at the final analysis, HERALD Trial CV-NCOV-004 willcontinue and remain observer blinded until the end of the trial {whenthe last subject has completed the last visit on Day 393 (see Section5.4)}. During this period, collection of placebo-controlled safety dataand accrual of COVID-19 cases will continue.

Extension Study

Following completion of the trial on Day 393, subjects will continueparticipating in the 1 year (12-month) extension of HERALD TrialCV-NCOV-004. During the Extension Study, blinding at the site level willbe maintained for the collection of additional placebo controlled datafor safety (SAEs), persistence of antibodies to SARS-CoV-2, andoccurrence of COVID-19 cases to assess duration of efficacy. TheExtension Study may be terminated upon approval of CVnCoV, at which timecontrol subjects may be offered vaccination with CVnCoV as soon asfeasible. The Extension Study may also be terminated upon deployment ofan effective vaccine locally. Before terminating the Extension Study,this will be discussed with the DSMB and Investigators as well as withthe relevant regulatory agencies.

5.2 Scientific Rationale for Trial Design

HERALD Trial CV-NCOV-004 will be conducted in 2 parts: an initial Phase2b trial followed by transition to a large Phase 3 efficacy trial. BothPhase 2 and Phase 3 parts of the trial are consistent in design, so thatcases of COVID-19 occurring in Phase 2 can be pooled with those in Phase3 for the primary analysis of VE. Combining COVID-19 cases in Phase 2and 3 to expedite an efficacy outcome was considered warranted in apandemic setting.

Both Phase 2b and Phase 3 will be randomized, observer-blinded, andplacebo-controlled. The difference in appearance and presentation of theinvestigational CVnCoV vaccine and placebo requires the trial to beconducted in an observer-blinded manner, which is a commonly used andwell-accepted method for trial blinding. The randomized, observerblinded, and placebo-controlled design will reduce the risk of bias inthe safety and efficacy outcomes of the trial (see also Section 7.3).

As the elderly are affected most by SARS CoV 2 and have a high risk forsevere disease and mortality, it is critical to investigate CVnCoV inthis population and therefore subjects≥61 years of age will be included.

The sample size of 4,000 subjects in Phase 2b is based on generating arobust and detailed dataset characterizing the safety, reactogenicity,and immunogenicity of CVnCoV prior to entering Phase 3. Furthermore, thedata generated in Phase 2b will be the main dataset to be submitted insupport of early conditional approval of CVnCoV. The total sample sizeof 36,500 subjects for the combined Phase 2b/3 trial is based ondemonstrating VE above 30% (based on a margin of 30% for the lower boundof the 97.5% CI for VE) when considering VE is 60%. With a 2-sided alphaof 2.5% and a total of 185 COVID-19 cases, the trial will have a 90%power to demonstrate a VE above 30%. Assuming an incidence rate ofCOVID-19 of 0.15% per month in control subjects; and a non-evaluablerate of 20% during the trial which includes 5% seropositivity ofenrollees at baseline (i.e. non-naïve subjects), follow-up of 36,500subjects enrolled over 3 months (18,250 per vaccine group) will accruethe target 185 COVID-19 cases approximately 9 months after the firstvaccination.

For the co-primary analyses of efficacy, COVID-19 case ascertainmentbegins at 15 days following the second vaccination of CVnCoV. This timepoint allows the immune response to mature and reach its full heightfollowing the second dose. As such, case ascertainment starting at thistime point represents the evaluation of full VE of CVnCoV against COVID19.

The safety objective of Phase 3 is to generate a large-scale safetydatabase that will demonstrate the safety of CVnCoV. All subjectsparticipating in the Phase 2b and Phase 3 parts of the trial will havemedically-attended AEs collected for 6 months after the secondvaccination; and AESIs and SAEs collected for 1 year after the secondvaccination. As such, each subject will participate in the trial forapproximately 13.5 months for the safety follow-up. Individuals withhistory of virologically-confirmed COVID-19 illness will be excludedfrom participating in this trial. However, this trial will not screenfor or exclude participants with history or laboratory evidence of priorSARS-CoV-2 infection, many of which might have been asymptomatic.Because pre-vaccination screening for prior infection is unlikely tooccur in practice, it is important to understand vaccine safety andCOVID-19 outcomes in in individuals with prior infection with SARS-CoV-2virus.

5.3 Justification for Dose Selection of the 12 μg mRNA dose level ofCVnCOV for Trial CV-NCOV-004 was based on the safety, tolerability andimmunogenicity results from Trials CV-NCOV-001 and CV NCOV-002.

5.4 End of Trial Definition

A subject is considered to have completed the trial when he/she hascompleted all visits, and procedures and tests applicable for the groupto which he/she was randomized to.

End of Trial CV-NCOV-004 is defined as when the last subject hascompleted the last visit on Day 393 or prematurely discontinued thetrial.

All subjects are expected to continue in the 1 year Extension Study inwhich the end of the trial is defined as when the last subject hascompleted the last visit on Day 757.

5.5 Stopping/Pausing Rules for Safety

5.5.1 Individual Subject Stopping Rules

The individual subject stopping rules are met in case any of thefollowing events occur after the first trial vaccination:

-   -   An allergic/anaphylactic reaction considered as related to the        trial vaccine    -   Any SAE considered as related to the trial vaccine

If any of these rules are met, the subject must not receive the secondvaccine dose. The subject will be encouraged to continue participationuntil the end of the trial for safety.

5.5.2 Pausing of the Trial

The decision to pause the trial (i.e. temporary stopping of enrollmentand vaccinations) due to a safety signal will be based on arecommendation from the DSMB in consultation with the Sponsor (seeSection 9.3.9.1). The DSMB may recommend pausing the trial for a safetyconcern following a review of accumulating safety data presented at theregularly scheduled DSMB meetings or from an ongoing review of AEs,which include but are not limited to, suspected unexpected seriousadverse reactions (SUSARs); all SAEs judged as related to trial vaccine;concerning SAEs (e.g., AESIs); and all life-threatening AEs and deaths.These events will be monitored by the DSMB on a regular basis during thetrial. The selected AEs and procedures for the safety review aredescribed in detail in the DSMB Charter.

To ensure subject safety on an ongoing basis, a blinded listing of theAEs as described above will be routinely monitored by the Chair of theDSMB (or designee) at regular intervals. For each review, the Chair {ordesignee(s)} will determine whether any single event or group of eventsconstitute a new safety signal. If not, the Chair will inform the StudyTeam that there are no safety concerns. Conversely, if there is a safetyconcern, the Chair may unblind the AE or AEs and, if necessary, convenean ad-hoc DSMB meeting for further assessment of the event(s).

Based on the assessment of the benefit-risk ratio and biologicplausibility of a causal relationship of the AE(s) to the trial vaccine,the DSMB will make a recommendation to the Sponsor to either continuethe trial as planned, modify its conduct, or pause the trial to allowfurther evaluation of the AE. If the latter, the Sponsor will make thedecision to pause the study in consultation with the DSMB.

Please refer to the DSMB Charter for additional discussion of the DSMB'srole and responsibilities.

6 Trial Population

The criteria for enrollment are to be followed explicitly. If it isnoted that a subject who does not meet one or more of the inclusioncriteria and/or meets one or more of the exclusion criteria isinadvertently enrolled and dosed, the Sponsor must be contactedimmediately.

In this trial, individuals with a history of virologically-confirmedCOVID-19 illness will be excluded from the trial. However, this trialwill not screen for or exclude individuals with a history or laboratoryevidence of prior SARS-CoV-2 infection. In addition, routine RT PCRtesting will not be performed at screening to exclude individuals withSARS CoV 2 infection at the time of enrollment. Any country specificregulation(s) will be adhered to in addition.

6.1 Inclusion Criteria for all Subjects

Subjects will be enrolled in this trial only if they meet all of thefollowing criteria:

1. Male or female subjects 18 years of age or older.

2. Provide written informed consent prior to initiation of any trialprocedures.

3. Expected compliance with protocol procedures and availability forclinical follow-up through the last planned visit.

4. Females of non-childbearing potential defined as follows: surgicallysterile (history of bilateral tubal ligation, bilateral oophorectomy orhysterectomy) or postmenopausal {defined as amenorrhea for 12consecutive months prior to screening (Day 1) without an alternativemedical cause}. A follicle-stimulating hormone (FSH) level may bemeasured at the discretion of the Investigator to confirm postmenopausalstatus.

5. Females of childbearing potential: negative urine pregnancy test{human chorionic gonatropin {hCG)}} within 24 hours prior to each trialvaccination on Day 1 and Day 29.

6. Females of childbearing potential must use highly effective methodsof birth control from 2 weeks before the first administration of thetrial vaccine until 3 months following the last administration. Thefollowing methods of birth control are considered highly effective whenused consistently and correctly:

-   -   Combined (estrogen and progestogen containing) hormonal        contraception associated with inhibition of ovulation (oral,        intravaginal or transdermal);    -   Progestogen-only hormonal contraception associated with        inhibition of ovulation (oral, injectable or implantable);    -   Intrauterine devices (IUDs);    -   Intrauterine hormone-releasing systems (IUSs);    -   Bilateral tubal occlusion;    -   Vasectomized partner or infertile partner;    -   Sexual abstinence {periodic abstinence (e.g., calendar,        ovulation, symptothermal and post-ovulation methods) and        withdrawal are not acceptable}.

6.2 Exclusion Criteria

Subjects will not be enrolled in this trial if they meet any of thefollowing criteria:

1. History of virologically-confirmed COVID-19 illness.

2. For females: pregnancy or lactation.

3. Use of any investigational or non-registered product (vaccine ordrug) within 28 days preceding the administration of the first trialvaccine or planned use during the trial.

4. Receipt of licensed vaccines within 28 days (for live vaccines) or 14days (for inactivated vaccines) prior to the administration of the firsttrial vaccine.

5. Prior administration of any investigational SARS-CoV-2 vaccine oranother coronavirus (SARS-CoV, MERS-CoV) vaccine or planned use duringthe trial.

6. Any treatment with immunosuppressants or other immune-modifying drugs(including but not limited to corticosteroids, biologicals andmethotrexate) for >14 days total within 6 months preceding theadministration of trial vaccine or planned use during the trial. Forcorticosteroid use, this means prednisone or equivalent, 0.5 mg/kg/dayfor 14 days or more. The use of inhaled, topical, or localizedinjections of corticosteroids (e.g., for joint pain/inflammation) ispermitted.

7. Any medically diagnosed or suspected immunosuppressive orimmunodeficient condition based on medical history and physicalexamination including known infection with human immunodeficiency virus(HIV), hepatitis B virus (HBV) or hepatitis C virus (HCV); currentdiagnosis of or treatment for cancer including leukemia, lymphoma,Hodgkin disease, multiple myeloma, or generalized malignancy; chronicrenal failure or nephrotic syndrome; and receipt of an organ or bonemarrow transplant.

8. History of angioedema (hereditary or idiopathic), or history of anyanaphylactic reaction or pIMD.

9. History of allergy to any component of CVnCoV vaccine.

10. Administration of immunoglobulins or any blood products within 3months prior to the administration of trial vaccine or planned receiptduring the trial.

11. Subjects with a significant acute or chronic medical or psychiatricillness that, in the opinion of the Investigator, precludes trialparticipation (e.g., may increase the risk of trial participation,render the subject unable to meet the requirements of the trial, or mayinterfere with the subject's trial evaluations). These include severeand/or uncontrolled cardiovascular disease, gastrointestinal disease,liver disease, renal disease, respiratory disease, endocrine disorder,and neurological and psychiatric illnesses. However, those withcontrolled and stable cases can be included in the trial.

12. Subjects with impaired coagulation or any bleeding disorder in whoman intramuscular injection or a blood draw is contraindicated.

13. Foreseeable non-compliance with the trial procedures as judged bythe Investigator. 6.3 Vaccine Delay Recommendations After enrollment,subjects may encounter clinical circumstances that could warrant a delayof trial vaccine administration as described below.

-   -   Subjects with a clinically significant Grade 2) active infection        or other acute disease (as assessed by the Investigator) or        temperature 38.0° C. 100.4° F.), within 3 days of intended trial        vaccination on Day 1 or Day 29. This includes symptoms that        could represent COVID-19 illness.        -   Trial vaccination should be delayed until the active            infection or other acute disease has recovered to Grade 1 or            the subject's temperature has decreased to <38.0° C.            (<100.4° F.). Following resolution of the illness, the            subject may be rescheduled for trial vaccination based on            the judgment of the Investigator.        -   Afebrile subjects with a minor illness may be vaccinated at            the discretion of the Investigator.    -   Receipt of a licensed vaccine within 28 days (for live vaccines)        or 14 days (for inactivated vaccines) prior to or after        scheduled administration of trial vaccine. As these are        recommended windows, rescheduling trial vaccination to be        compliant with these windows should only be done if practical.

6.4 Failure to Meet Eligibility Criteria

The Investigator must account for all subjects who sign an informedconsent. If the subject is found to be not eligible (i.e., did not meetall inclusion criteria or met one or more exclusion criteria), theInvestigator should document this in the subject's source documents.

7 Trial Vaccine

7.1 Trial Vaccine Administration

7.1.1 Description of the Trial Vaccines

CVnCoV is an investigational LNP-formulated RNActive® SARS-CoV-2vaccine. The IMP is composed of the active pharmaceutical ingredient, anmRNA that encodes Wsmpv-SP, and 4 lipid components: cholesterol, 1,2distearoyl-sn-glycero-3-phosphocholine (DSPC), PEG-ylated lipid and acationic lipid. It is supplied as a concentrate at 1 mg/mL of mRNA drugsubstance.

The placebo vaccine will be sterile normal saline (0.9% NaCl) forinjection.

7.1.2 Dosing and Administration

7.1.2.1 CVnCoV

Subjects randomized to CVnCoV will receive 2 injections of CVnCoV at adose level of 12 μg mRNA, administered 28 days apart.

Administration of CVnCoV must be performed by intramuscular (IM)injection in the deltoid area, preferably in the non-dominant arm.CVnCoV is intended strictly for IM injection and must not be injectedsubcutaneously, intradermally, or intravenously. The instructions forinjection as described in the Pharmacy Manual must be followed.

7.1.2.2 Placebo Control (Normal Saline)

Subjects randomized to the control arm of the trial will receive 2 dosesof saline placebo {normal saline (0.9% NaCl) for injection},administered 28 days apart.

Administration of saline placebo must be performed by IM injection inthe deltoid area, preferably in the non-dominant arm. The instructionsfor injection described in the Pharmacy Manual must be followed.

7.1.2.3 Hypersensitivity Reactions to Vaccination

CVnCoV should not be administered to subjects with a knownhypersensitivity to any of the components of the vaccine.

Since there is a theoretical risk of anaphylactic reactions, trialvaccine must only be administered if emergency equipment for thetreatment of anaphylactic reactions (intravenous fluids,corticosteroids, H1 and H2 blocking agents, epinephrine, equipment forcardiopulmonary resuscitation) is readily available. All subjects mustremain under direct supervision of personnel trained in the treatment ofthese reactions for at least 30 minutes following administration oftrial vaccine.

If anaphylaxis or severe hypersensitivity reactions occur followingtrial vaccine administration, no further doses should be given (seeSections 5.5.1 and 8.1).

7.2 Preparation/Handling/Storage/Accountability

Refer to the Pharmacy Manual for detailed information on thepreparation, handling, storage and blinding of CVnCoV and salineplacebo.

7.2.1 CVnCoV Preparation

The concentrated CVnCoV must be diluted in the provided sterile normalsaline (0.9% NaCl) diluent containing preservative to produce the dosesolution for IM injection. This will be prepared by an unblindedqualified pharmacist according to the Handling Manual for the IMPprovided by CureVac AG. The pharmacist will have no other trial functionfollowing vaccination and will maintain the treatment assignments instrict confidence.

7.2.2 CVnCoV Product Storage and Stability

Concentrated CVnCoV will be shipped to the site frozen at below −60° C.

Once at the site, concentrated CVnCoV should be stored frozen at below−60° C.

7.2.3 Placebo Control (Normal Saline)

The normal saline placebo control vaccine should be stored according tothe Summary of Product Characteristics.

7.2.4 Accountability

It is the responsibility of the Investigator to ensure that the currentand accurate records of trial supplies received, stored, and dispensedat the site are maintained using appropriate forms according toapplicable regulations and guidelines. The trial supplies must be storedunder the recommended storage conditions, locked with restricted access(refer to the Pharmacy Manual). Authorized personnel must dispense thevaccine at the trial site and in accordance with the protocol andapplicable regulations and guidelines.

IMP accountability and inventory logs must be kept up-to-date at thetrial site with the following information:

-   -   Dates and quantities of CVnCoV received from CureVac.    -   Unique subject identifier.    -   Date and quantity of trial vaccine dispensed to each subject.    -   Initials of the person preparing the dose.    -   Initials of the person administering the vaccine.

These logs must be readily available for inspections and are open toregulatory inspection at any time.

7.3 Randomization and Blinding

Both Phase 2b and Phase 3 will be randomized, observer-blinded, andplacebo-controlled. The difference in appearance of the investigationalCVnCoV vaccine and placebo required the trial to be conducted in anobserver-blinded manner, which is a well-accepted method for blinding.

7.3.1 Randomization

Subjects 18 years of age or older will be enrolled at multiple sitesglobally and will be randomized in a 1:1 ratio to receive either CVnCoVor placebo. The randomization will be performed centrally and stratifiedby country and age group (18 to 60 and ≥61 years of age). Therandomization scheme will be generated and maintained by an IndependentStatistical group at the contract research organization (CRO), PRA.Subjects will be enrolled into the trial online and randomized using aninteractive web response system (IWRS). After demographic andeligibility criteria are entered into the system, each subject enrolledinto the trial will be assigned their treatment assignment.

7.3.2 Blinding

Subjects will be randomized and vaccinated with CVnCoV or placebo in anobserver blinded manner (due to the difference in appearance andpresentation of the investigational CVnCoV vaccine and placebo). Thepharmacist at the site will not be blinded to the identity of the trialvaccine being administered to the subject. However, the vaccinator,Investigator and all site personnel involved in the conduct of the trial(including follow-up of safety and COVID-19 case ascertainment) will beblinded to trial vaccine and subject treatment assignments. To maintainthe blinding of the vaccinator, the pharmacist will provide the dose oftrial vaccine to the vaccinator prefilled in a syringe with a labelcovering the liquid contents so that it is not visible. All personnel atthe CRO and Sponsor directly involved in the conduct of the trial willalso be blinded. There will be certain individuals at the CRO andSponsor whose function requires them to be unblinded during the trial{e.g., unblinded monitoring for trial vaccine accountability in thepharmacy; unblinded independent statistician assisting the DSMB; reviewof immunogenicity data (see next paragraph)}. These unblindedindividuals will be identified and their responsibilities documented.Because the immunogenicity results would unblind the subject's treatmentassignment, the independent laboratory performing the assays will keepthe results in strict confidence. An unblinded person, named at thestart of the trial and independent of the conduct of the trial, willhave the responsibility of reviewing the quality of the immunogenicitydata as it is being generated. This person will maintain the results instrict confidence. To maintain the blind, the immunogenicity data willonly be merged with the clinical database following unblinding of thetrial.

It will be at the discretion of the DSMB members whether or not safetydata reviewed at the DSMB meetings will be unblinded. If there are anysafety concerns, the DSMB may request unblinding of an individualsubject or a specific dataset at any time. In addition, the DSMB willperiodically monitor COVID-19 cases by vaccine group for signals of VDE.At the interim analyses, the DSMB will review cases of COVID-19 cases byvaccine group for efficacy or futility, and will communicate the outcometo the Sponsor in a blinded manner.

For the submission of documents for regulatory approval during theongoing conduct of Trial CV-NCOV-004 (e.g., if efficacy is demonstratedat one of the interim analyses), an unblinded Submission Team will beformed which will be completely independent of the team conducting thetrial. The Submission Team will comprise individuals from the Sponsorand CRO, and their roles and responsibilities on the unblinded team willbe clearly defined.

7.3.3 Emergency Unblinding

Individual unblinding should only occur in emergency situations forreasons of subject safety when knowledge of the trial vaccine isessential for the clinical management or welfare of the subject.Unblinding in this situation will be based on the judgment of theInvestigator, ideally in discussion with the Sponsor.

In general, the identity of the trial vaccine should not affect theclinical management of any SAE/AE. Whenever possible, the Investigatorshould attempt to contact the Sponsor before breaking the blind todiscuss the need for emergency unblinding. Once agreed, code-breakingwill be carried out via the IWRS.

When the blind is broken, the date, exact timing, and reason must befully documented in the source documents. The Investigator should notinform other blinded trial staff of the identity of the IMP.

If the code has been broken and there are no medical reasons fordiscontinuation, the subject may continue in the trial. If the subjecthas received at least 1 dose of trial vaccine, it will be the judgmentof the Investigator, in consultation with the Sponsor, whether thesubject will be vaccinated with the second dose. If the subject isdiscontinued from the trial, every effort should be made to continuesafety follow-up of the subject until the end of the trial.

7.4 Vaccine Compliance

The Investigator must record all trial vaccinations administered in thesubject's eCRF page.

7.5 Misuse and Overdose

Definition of misuse: Situations where the trial vaccine isintentionally and inappropriately used not in accordance with theprotocol dosing instructions or authorized product information.

Definition of overdose: Administration of a quantity of the trialvaccine given per administration or cumulatively which is above themaximum recommended dose according to the protocol dosing instructionsor authorized product information.

No toxic effects are expected from current clinical and non-clinicalexperience. Possible local reactions (pain) or systemic AEs (fever,headache, fatigue, chills, myalgia, arthralgia, nausea/vomiting anddiarrhea) may be treated symptomatically with physical measures,paracetamol, or non-steroidal anti-inflammatory drugs.

7.6 Concomitant Therapy and Vaccines

Concomitant medication and vaccines including the reason foradministration must be recorded in the subject's eCRF.

7.6.1 Permitted Medications/Vaccines During the Trial

Subjects are permitted to use antipyretics and other pain medications totreat any ongoing condition(s) the subject may have. Antipyretics (e.g.,paracetamol) or other pain medication may be used to treat any localand/or systemic reactions associated with trial vaccination. Paracetamoltaken prophylactically for potential vaccine-associated reactions isalso permitted in this trial. For example, if a subject experiencesadverse reactions following the first trial vaccination, paracetamol maybe taken prophylactically for these reactions for the second trialvaccination. In this case, paracetamol (up to 1 gram dose) may be takenafter trial vaccination and at bedtime, and then in the morning and atbedtime during the next day. Alternatively, a 500 mg dose of paracetamolmay be taken every 6 hours after trial vaccination for up to 36 hours.The dose and dosing schedule of paracetamol should be discussed with theInvestigator.

Paracetamol administered as a treatment for vaccine-associated reactionsor for prophylaxis, along with timing of administration with respect totrial vaccination must be documented in the eCRF.

Other than the prohibited medications and vaccines described in Section6.2 and listed below in Section 7.6.2, medications that are required forthe treatment of the subject's pre existing medical conditions arepermitted.

7.6.2 Prohibited Medications/Vaccines During the Trial

-   -   Use of any investigational or non-registered product (vaccine or        drug) is prohibited during the trial.    -   Licensed vaccines should not be administered within 28 days (for        live vaccines) or 14 days (for inactivated vaccines) of trial        vaccine administration during the trial.    -   Receipt of any other investigational SARS-CoV-2 vaccine or other        coronavirus vaccine is prohibited during the trial.    -   Any treatment with immunosuppressants or other immune-modifying        drugs (including but not limited to corticosteroids, biologicals        and methotrexate) is prohibited during the trial. For        corticosteroid use, this means prednisone or equivalent, 0.5        mg/kg/day for 14 days or more. The use of inhaled, topical, or        localized injections of corticosteroids (e.g., for joint        pain/inflammation) is permitted.    -   Administration of immunoglobulins or any blood products is        prohibited during the trial.

7.7 Therapy Leading to Discontinuation

If a subject requires therapy listed as an exclusion criterion inSection 6.2 and which cannot be delayed, discontinuation would beconsidered to ensure integrity of the trial data, following individualcase review. Every effort should be made to continue safety follow-up ofthe subject until the end of the trial.

7.8 Treatment After the End of Trial

No post-trial care will be provided.

8 Discontinuation/Withdrawal Criteria

Participation in the trial is strictly voluntary. A subject has theright to withdraw from the trial at any time and for any reason. TheInvestigator has the right to withdraw a subject from further trialvaccine administration and/or the trial if this is considered in thesubject's best interest or as a result of a protocol deviation.

For discontinuations due to an AE, every effort should be made todocument the outcome of the event.

Subjects who received at least 1 dose of trial vaccine will beencouraged to continue participation until the end of the trial forsafety assessments.

8.1 Discontinuation of Trial Vaccine Administration

The primary reason for discontinuation of further administration oftrial vaccine will be recorded in the subject's eCRF according to thefollowing categories:

-   -   Consent withdrawal by the subject.

The reason for withdrawal, if provided, should be recorded in the eCRF.

Note: All attempts should be made to determine the underlying reason forthe withdrawal and, where possible, the primary underlying reason shouldbe recorded (i.e., withdrawal due to an AE should not be recorded in the“voluntary withdrawal” category).

-   -   AE (including known side effects of the trial vaccine).

If discontinuation is due to an AE possibly related to the trial vaccineor trial procedures, the subject must be followed-up by additionalexaminations according to the medical judgment of the Investigator untilthe condition is resolved or the Investigator deems further observationsor examinations to be no longer medically indicated.

-   -   Change in the subject's overall medical status prohibiting        further participation.    -   Pregnancy (see Section 9.3.5).

Any subject who, despite the requirement for adequate contraception,becomes pregnant during the trial will not receive further trial vaccinedoses. The site should maintain contact with the pregnant subject andcomplete a “Clinical Trial Pregnancy Form” as soon as possible. Inaddition, the subject should be followed-up until the birth of thechild, or spontaneous or voluntary termination. When pregnancy outcomeinformation becomes available, the information should be captured usingthe same form. The subject should be reported as an IMP discontinuationand the reason (i.e. pregnancy) should be given.

-   -   Trial terminated by the Sponsor (in which case the minimum        safety follow-up conducted at the end of trial visit on Day 393        would be performed).    -   Major protocol deviation.    -   Other.

Note: The specific reasons should be recorded in the “specify” field ofthe eCRF.

8.2 Withdrawal from the Trial

Subjects should be withdrawn from the trial in case any of the followingsituations occur:

-   -   Continued participation jeopardizes the subject's health,        safety, or rights.    -   The subject has experienced an AE that requires early        termination because continued participation imposes an        unacceptable risk to the subject's health or the subject is        unwilling to continue because of the AE. The reasons for not        performing further safety or immunogenicity assessments should        be documented.    -   The subject did not return to the site and multiple attempts (a        minimum of 3 attempts) to contact the subject were unsuccessful        (lost to follow-up).    -   The subject wishes to withdraw from the trial. The reason for        withdrawal, if provided, should be recorded. All attempts should        be made to determine the underlying reason for the withdrawal        and, where possible, the primary underlying reason should be        recorded (i.e., withdrawal due to an AE should not be recorded        in the “voluntary withdrawal” category).

Any subject who prematurely terminates participation and who hasreceived at least one trial vaccine dose will undergo the sameprocedures as for the end of trial visit, unless such procedures areconsidered to pose unacceptable risk to the subject.

Discontinued or withdrawn subjects will not be replaced.

8.3 Trial Termination

The Sponsor reserves the right to terminate the trial at any time.Possible reasons for trial termination include the following:

-   -   Outcome of the interim analysis may show high VE or futility.    -   Safety reasons: the incidence of AEs in this or any other trial        using a related vaccine indicates a potential health risk for        the subjects.    -   New scientific knowledge becomes known that makes the objectives        of the trial no longer feasible/valid.    -   The site is unlikely to be able to recruit sufficient subjects        within the agreed time frame.    -   The site does not respond to trial management requests.    -   Repeated protocol deviations.    -   Unsafe or unethical practices.    -   Administrative decision.

Following a trial termination decision, the Investigator must contactall subjects within a time period set by the Sponsor. All trialmaterials must be collected and relevant documentation completed to thegreatest extent possible. The trial can also be terminated by theRegulatory Authority for any reason or if recommended by the DSMB, or ata site level by the Independent Ethics Committee or Institutional ReviewBoard (IEC/IRB). The Sponsor may close an individual site prematurelyfor reasons such as poor protocol compliance or unsatisfactoryrecruitment of subjects.

8.4 Lost to Follow-Up

All efforts should be made to contact subjects who have not returned forthe scheduled trial visit or who are unable to be contacted for ascheduled phone call. A minimum of 3 attempts should be made anddocumented. If a subject is lost to follow-up before resolution ofrelated SAEs or AEs, the Sponsor may consider further attempts tocontact the subject in order to collect follow-up safety information.

9 Trial Assessments and Procedures

The trial assessments and procedures are discussed in this section.

For subjects who are unable to come to the site for protocol-specifiedsite visits (e.g., due to the public health emergency related toCOVID-19), safety assessments may be performed using alternative methods(e.g., phone contact, virtual visit, alternative location forassessment).

For further flexibility in trial conduct in the pandemic setting, homevisits will be allowed to perform safety assessments and proceduresincluding the collection of blood and any bio samples. If site visits,phone contacts or sample collection cannot be performed within theprotocol defined windows, in such unique circumstances as a publichealth emergency, it will be acceptable to perform these tasks outsideof these windows. In the pandemic setting, the protocol-defined windowsfor site visits and phone contacts are provided for guidance and willnot be considered deviations, if not strictly adhered to.

An electronic diary (eDiary) will be used during the trial for efficientcollection of safety related information. However, paper diaries may besubstituted for some subjects during the trial.

Initiation of subject enrollment of the 2 target age groups into Phase2b will be flexible. Depending on the timing of data from the Phase 1and Phase 2a trials, enrollment of the 2 age groups into Phase 2b may bestaggered, initially starting with subjects 18 to 60 years of agefollowed by subjects≥61 years of age. As the older age group willcomprise 20% to 25% of the total number of subjects in Phase 2b, thisstaggered start is not expected to impact overall enrollment of thePhase 2b cohort.

9.1 Schedule of Trial Assessments and Procedures

By signing the informed consent form, subjects will be consenting toparticipate in both Trial CV-NCOV-004 and its 1 year Extension Study fora total of approximately 2.1 years of participation.

The trial assessments and procedures apply to all subjects, independentif they had known SARS CoV-2 positive serology before the trial orindependent of the serology status at baseline as per retrospectiveanalysis.

Subjects participating in Phase 2b will be given a thermometer tomeasure body temperature orally and a measuring tape to determine thesize of local injection site reactions. Subjects will be instructed onhow to enter the solicited AEs daily for 7 days in the eDiary.

During the conduct of the trial and interactions with subjects, anyperson with early warning signs of COVID-19 should be referred toemergency medical care immediately. These signs include, but are notlimited to, the following: difficulty breathing, persistent pain orpressure in the chest, new confusion, inability to awake or stay awake,or bluish lips or face.

9.1.1 Phase 2b: Immunogenicity Subset

The Immunogenicity Subset of Phase 2b will include the first 600subjects enrolled into each of the 2 age groups, 18-60 and ≥61 years ofage, into Phase 2b. As such, the target total enrollment will beapproximately 1,200 subjects.

9.1.1.1 Clinic Visit 1: Day 1—First Trial Vaccination

Note that procedures to establish subject eligibility, recording ofdemographic information and medical history may be performed within 21days prior to trial vaccine administration, i.e., spread out over morethan 1 day. However, if all information is available and assessments andprocedures can be performed, eligibility can be established on the sameday of trial vaccine administration. All eligibility criteria must bereviewed prior to trial vaccine administration on Day 1.

Pre-Vaccination Procedures

-   -   Obtain signed informed consent form.    -   Signed informed consent must be obtained prior to the subject        entering into the trial, and before any protocol-directed        procedures are performed.    -   By signing the informed consent form, the subject voluntarily        agrees to participate in the HERALD Trial CV-NCOV-004 and its 1        year Extension Study for a total of approximately 2 years.    -   Review inclusion/exclusion criteria (see Section 6.1 and 6.2)        and review prohibited medications listed as an exclusion        criterion (see Section 6.2).    -   Record demographic information.    -   Record medical history.    -   Record concomitant medications and vaccinations, including        recurring medications for intermittent conditions, if taken        within 6 months prior to enrollment in this trial.    -   Perform a complete physical examination, including height and        weight (see Section 9.3.7). If the complete physical examination        to establish eligibility was performed within 21 days prior to        trial vaccine administration, a symptom-directed physical        examination should be performed on the day of vaccination prior        to trial vaccine administration.    -   Measure vital signs (body temperature, pulse, blood pressure;        see Section 9.3.7).    -   Perform urine pregnancy test in females of childbearing        potential.    -   Collect pre-vaccination blood samples for binding antibody        testing to RBD of S protein of SARS-CoV-2 (˜6 mL blood);        SARS-CoV-2 viral neutralizing activity (˜6 mL blood); and        binding antibody testing to N protein of SARS CoV 2 (˜6 mL        blood).    -   Collect pre-vaccination blood samples for genomic biomarkers (˜6        mL blood) from subjects at selected site(s).    -   Collect pre-vaccination blood samples for CMI (˜32 mL blood)        from subjects at selected site(s).

Vaccination Procedure

-   -   Review criteria for delay or cancellation of vaccination. See        Sections 6.3 and 8.1 for an overview of the criteria leading to        delay or cancellation of vaccine administration. In case of        delay, the vaccination should take place within the allowed time        windows. The reasons for delay or cancellation should be        documented in the subject's chart.    -   Administer the trial vaccine dose according to the subject's        assignment.

Post-Vaccination Procedures

-   -   Observe the subject on site for at least 30 minutes following        vaccination for safety monitoring. At the end of the observation        period:    -   Measure vital signs (body temperature, pulse, blood pressure;        see Section 9.3.7).    -   The subject may not be discharged until vital signs are within        normal range or have returned to pre-vaccination levels.    -   Record the occurrence of any AEs following trial vaccination.    -   Instructions for the subject:    -   Instruct the subject how to measure solicited AEs and how to        complete the eDiary. The subject should record solicited local        and systemic AEs occurring on the day of vaccination and the        following 7 days, and unsolicited AEs (i.e., the occurrence of        all other AEs) occurring on the day of vaccination and the        following 28 days.    -   Remind the subject to call the site immediately to report the        following:    -   If he/she experiences any concerning local or systemic reactions        or other medical event.    -   Any medically-attended visits that are not routine visits for        physical examination or vaccination, such as visits for        hospitalization, an emergency room visit, or an otherwise        unscheduled visit to or from medical personnel (medical doctor)        for any reason.    -   Experience a serious medical event, have a change in overall        health or be diagnosed with a new medical condition by a doctor.        These should be reported regardless of the perceived        relationship between the event and the trial vaccine.    -   Remind the subject to contact the site immediately if he/she has        any of the symptoms suggestive of COVID-19. In addition,        subjects will be messaged up to twice a week to provide a yes or        no response to having COVID-19 symptoms. Those who respond “yes”        will be contacted by trial staff for follow-up information (see        Section 9.2.1 and Section 9.5).    -   The subject should also be reminded to contact the site        immediately if he/she had a positive SARS-CoV-2 test performed        outside of the site, whether they were symptomatic (COVID-19        illness) or asymptomatic at the time of the test.

Note: Subjects without symptoms may have been tested for severalreasons, for example, close exposure to a known person with SARS-CoV-2infection or as part of their routine screening as a healthcareprovider.

9.1.1.2 Phone Call: Day 2 (−0/+0 day)

The purpose of this phone contact is to inquire about the subject'sgeneral well-being and to assess safety 1 day after the first trialvaccination.

-   -   During the phone call:    -   Review and record any newly reported safety data including        solicited and unsolicited AEs, or other AEs (medically-attended        AEs, SAEs).    -   Record concomitant medications and vaccinations, including        recurring medications for intermittent conditions.

If the subject reports any concerning local or systemic reactions, orother AEs (e.g., medically-attended AEs, SAEs), these should befollowed-up either by a phone call(s) or by an unscheduled site visitbased on the judgment of the Investigator.

-   -   Instructions for the subject:    -   Remind the subject to continue recording solicited and        unsolicited AEs (i.e., the occurrence of all other AEs) in the        eDiary.

Remind the subject to call the site immediately to report the following:

-   -   If he/she experiences any concerning local or systemic reactions        or other medical event.    -   Any medically-attended visits that are not routine visits for        physical examination or vaccination, such as visits for        hospitalization, an emergency room visit, or an otherwise        unscheduled visit to or from medical personnel (medical doctor)        for any reason.    -   Experience a serious medical event, have a change in overall        health or be diagnosed with a new medical condition by a doctor.        These should be reported regardless of the perceived        relationship between the event and the trial vaccine.    -   Remind the subject to contact the site immediately if he/she has        any of the symptoms suggestive of COVID-19. In addition,        subjects will be messaged up to twice a week to provide a yes or        no response to having COVID-19 symptoms. Those who respond “yes”        will be contacted by trial staff for follow-up information (see        Section 9.2.1 and Section 9.5).    -   The subject should also be reminded to contact the site        immediately if he/she had a positive SARS-CoV-2 test performed        outside of the site, whether they were symptomatic (COVID-19        illness) or asymptomatic at the time of the test.

9.1.1.3 Clinic Visit 2: Day 29—Second Trial Vaccination (−3/+7 days)

Pre-Vaccination Procedures

-   -   Review and record any newly reported safety data including        solicited and unsolicited AEs, or other AEs (medically-attended        AEs, SAEs).    -   Record concomitant medications and vaccinations, including        recurring medications for intermittent conditions.    -   Perform a symptom-directed physical examination (see Section        9.3.7).    -   Measure vital signs (body temperature, pulse, blood pressure,        see Section 9.3.7).    -   Perform urine pregnancy test in females of childbearing        potential.    -   Collect pre-vaccination blood samples for binding antibody        testing to RBD of S protein of SARS-CoV-2 (˜6 mL blood) and        SARS-CoV-2 viral neutralizing activity (˜6 mL blood). No testing        of antibody to N protein of SARS-CoV-2 will performed at this        time point.    -   Collect pre-vaccination blood samples for genomic biomarkers (˜6        mL blood) from subjects at selected site(s).    -   Collect pre-vaccination blood samples for CMI (˜32 mL blood)        from subjects at selected site(s).

Vaccination Procedure

-   -   Review criteria for delay or cancellation of vaccination. See        Sections 6.3 and 8.1 for an overview of the criteria leading to        delay or cancellation of vaccine administration. In case of        delay, the vaccination should take place within the allowed time        windows. The reasons for delay or cancellation should be        documented in the subject's chart.    -   Administer the trial vaccine dose according to the subject's        assignment.

Post-Vaccination Procedures

-   -   Observe the subject on site for at least 30 minutes following        vaccination for safety monitoring. At the end of the observation        period:    -   Measure vital signs (body temperature, pulse, blood pressure;        see Section 9.3.7).    -   The subject may not be discharged until vital signs are within        normal range or have returned to pre-vaccination levels.    -   Record the occurrence of any AEs following trial vaccination.    -   Instructions for the subject:    -   Re-instruct the subject how to measure solicited AEs and how to        complete the eDiary. The subject should record solicited local        and systemic AEs occurring on the day of vaccination and the        following 7 days, and unsolicited AEs (i.e., the occurrence of        all other AEs) occurring on the day of vaccination and the        following 28 days.    -   Remind the subject to call the site immediately to report the        following:    -   If he/she experiences any concerning local or systemic reactions        or other medical event.    -   Any medically-attended visits that are not routine visits for        physical examination or vaccination, such as visits for        hospitalization, an emergency room visit, or an otherwise        unscheduled visit to or from medical personnel (medical doctor)        for any reason.    -   Experience a serious medical event, have a change in overall        health or be diagnosed with a new medical condition by a doctor.        These should be reported regardless of the perceived        relationship between the event and the trial vaccine.    -   Remind the subject to contact the site immediately if he/she has        any of the symptoms suggestive of COVID-19. In addition,        subjects will be messaged up to twice a week to provide a yes or        no response to having COVID-19 symptoms. Those who respond “yes”        will be contacted by trial staff for follow-up information (see        Section 9.2.1 and Section 9.5).    -   The subject should also be reminded to contact the site        immediately if he/she had a positive SARS-CoV-2 test performed        outside of the site, whether they were symptomatic (COVID-19        illness) or asymptomatic at the time of the test.

9.1.1.4 Phone Call: Day 30 (−0/+0 day)

The purpose of this phone contact is to inquire about the subject'sgeneral well-being and to assess safety 1 day after the second trialvaccination.

The assessments and procedures are identical to those performed duringthe phone call on Day 2.

9.1.1.5 Clinic Visit 3: Day 43 (−3/+3 days)

-   -   Review and record any newly reported safety data including        solicited and unsolicited AEs, or other AEs (medically-attended        AEs, SAEs).    -   Record concomitant medications and vaccinations, including        recurring medications for intermittent conditions.    -   Perform a symptom-directed physical examination (see Section        9.3.7).    -   Measure vital signs (body temperature, pulse, blood pressure,        see Section 9.3.7).    -   Collect blood samples for binding antibody testing to RBD of S        protein of SARS CoV-2 (˜6 mL blood); SARS-CoV-2 viral        neutralizing activity (˜6 mL blood); and binding antibody        testing to N protein of SARS-CoV-2 (˜6 mL blood).    -   Collect blood samples for genomic biomarkers (˜6 mL blood) from        subjects at selected site(s).    -   Collect blood samples for CMI (˜32 mL blood) from subjects at        selected site(s).    -   Instructions for the subject:    -   Inform the subject that recording of solicited local and        systemic reactions in the eDiary is complete. Remind the subject        to continue recording unsolicited AEs (all AEs).    -   Remind the subject to call the site immediately to report the        following:    -   If he/she experiences any concerning medical event.    -   Any medically-attended visits that are not routine visits for        physical examination or vaccination, such as visits for        hospitalization, an emergency room visit, or an otherwise        unscheduled visit to or from medical personnel (medical doctor)        for any reason.    -   Experience a serious medical event, have a change in overall        health or be diagnosed with a new medical condition by a doctor.        These should be reported, regardless of the perceived        relationship between the event and the trial vaccine.    -   Remind the subject to contact the site immediately if he/she has        any of the symptoms suggestive of COVID-19. In addition,        subjects will be messaged up to twice a week to provide a yes or        no response to having COVID-19 symptoms. Those who respond “yes”        will be contacted by trial staff for follow-up information (see        Section 9.2.1 and Section 9.5).    -   The subject should also be reminded to contact the site        immediately if he/she had a positive SARS-CoV-2 test performed        outside of the site, whether they were symptomatic (COVID-19        illness) or asymptomatic at the time of the test.

9.1.1.6 Clinic Visit 4: Day 57 (−3/+7 days)

-   -   Review and record any newly reported safety data including        unsolicited AEs or other AEs (medically-attended AEs, SAEs).    -   Record concomitant medications and vaccinations, including        recurring medications for intermittent conditions.    -   Perform a symptom-directed physical examination (see Section        9.3.7).    -   Measure vital signs (body temperature, pulse, blood pressure,        see Section 9.3.7).    -   Collect a blood sample for immunogenicity assessment for binding        antibody testing to RBD of S protein of SARS-CoV-2 (˜6 mL blood)        and SARS-CoV-2 viral neutralizing activity (˜6 mL blood). (No        testing of binding antibody to N protein of SARS CoV 2 will        performed at this time point).    -   Instructions for the subject:    -   Inform the subject that reporting of unsolicited AEs is        complete.    -   Remind the subject to call the site immediately to report the        following:    -   If he/she experiences any concerning medical event.    -   Any medically-attended visits that are not routine visits for        physical examination or vaccination, such as visits for        hospitalization, an emergency room visit, or an otherwise        unscheduled visit to or from medical personnel (medical doctor)        for any reason.    -   Experience a serious medical event, have a change in overall        health or be diagnosed with a new medical condition by a doctor.        These should be reported regardless of the perceived        relationship between the event and the trial vaccine.    -   Remind the subject to contact the site immediately if he/she has        any of the symptoms suggestive of COVID-19. In addition,        subjects will be messaged up to twice a week to provide a yes or        no response to having COVID-19 symptoms. Those who respond “yes”        will be contacted by trial staff for follow-up information (see        Section 9.2.1 and Section 9.5).    -   The subject should also be reminded to contact the site        immediately if he/she had a positive SARS-CoV-2 test performed        outside of the site, whether they were symptomatic (COVID-19        illness) or asymptomatic at the time of the test.

9.1.1.7 Clinic Visit 5: Day 120 (−7/+7 days)

-   -   Review and record any newly reported AEs since the site visit on        Day 57 (medically-attended AEs, SAEs).    -   Record concomitant medications and vaccinations, including        recurring medications for intermittent conditions.    -   Perform a symptom-directed physical examination (see Section        9.3.7).    -   Measure vital signs (body temperature, pulse, blood pressure,        see Section 9.3.7).    -   Collect blood samples for binding antibody testing to RBD of S        protein of SARS CoV-2 (˜6 mL blood) and SARS-CoV-2 viral        neutralizing activity (˜6 mL blood). (No testing of binding        antibody to N protein of SARS CoV 2 will performed at this time        point).    -   Collect blood samples for genomic biomarkers (˜6 mL blood) from        subjects at selected site(s).    -   Collect blood samples for CMI (˜32 mL blood) from subjects at        selected site(s).    -   Instructions for the subject:    -   Remind the subject to call the site immediately to report the        following:    -   If he/she experiences any concerning medical event.    -   Any medically-attended visits that are not routine visits for        physical examination or vaccination, such as visits for        hospitalization, an emergency room visit, or an otherwise        unscheduled visit to or from medical personnel (medical doctor)        for any reason.    -   Experience a serious medical event, have a change in overall        health or be diagnosed with a new medical condition by a doctor.        These should be reported regardless of the perceived        relationship between the event and the trial vaccine.    -   Remind the subject to contact the site immediately if he/she has        any of the symptoms suggestive of COVID-19. In addition,        subjects will be messaged up to twice a week to provide a yes or        no response to having COVID-19 symptoms. Those who respond “yes”        will be contacted by trial staff for follow-up information (see        Section 9.2.1 and Section 9.5).    -   The subject should also be reminded to contact the site        immediately if he/she had a positive SARS-CoV-2 test performed        outside of the site, whether they were symptomatic (COVID-19        illness) or asymptomatic at the time of the test.

9.1.1.8 Clinic Visit 6: Day 211 (−7/+7 days)

The assessments and procedures are identical to those performed duringClinic Visit 5 on Day 120, except for the below.

-   -   Collect blood samples for binding antibody testing to RBD of S        protein of SARS CoV-2 (˜6 mL blood); SARS-CoV-2 viral        neutralizing activity (˜6 mL blood); and binding antibody        testing to N (nucleocapsid) protein of SARS-CoV-2 (˜6 mL blood).    -   Collect blood samples for genomic biomarkers (˜6 mL blood) from        subjects at selected site(s).

9.1.1.9 Phone Call: Day 302 (−7/+7 days)

The purpose of this phone contact is to inquire about the subject'sgeneral well-being and to assess safety since the site visit on Day 211.

-   -   During the phone call:    -   Review and record any newly reported AEs since the site visit on        Day 211 (SAEs).    -   Record concomitant medications and vaccinations, including        recurring medications for intermittent conditions.    -   If the subject reports by phone any concerning AEs, these should        be followed-up either by a phone call(s) or by an unscheduled        site visit based on the judgment of the investigator.    -   Instructions for the subject:    -   Remind the subject to call the site immediately to report the        following:    -   If he/she experiences any concerning medical event.    -   Any medically-attended visits that are not routine visits for        physical examination or vaccination, such as visits for        hospitalization, an emergency room visit, or an otherwise        unscheduled visit to or from medical personnel (medical doctor)        for any reason.    -   Experience a serious medical event, have a change in overall        health or be diagnosed with a new medical condition by a doctor.        These should be reported regardless of the perceived        relationship between the event and the trial vaccine.    -   Remind the subject to contact the site immediately if he/she has        any of the symptoms suggestive of COVID-19. In addition,        subjects will be messaged up to twice a week to provide a yes or        no response to having COVID-19 symptoms. Those who respond “yes”        will be contacted by trial staff for follow-up information (see        Section 9.2.1 and Section 9.5).    -   The subject should also be reminded to contact the site        immediately if he/she had a positive SARS-CoV-2 test performed        outside of the site, whether they were symptomatic (COVID-19        illness) or asymptomatic at the time of the test.

9.1.1.10 End of Trial Visit: Day 393 (−0/+21 days)

The end of trial visit will be performed on Day 393, 1 year after thelast trial vaccine administration. If possible, this visit shouldinclude subjects who prematurely discontinued vaccination during thetrial. The following assessments should be performed:

-   -   Review and record any newly reported AEs since the phone contact        on Day 302 (SAEs).    -   Record concomitant medications and vaccinations, including        recurring medications for intermittent conditions.    -   Perform a complete physical examination, including height and        weight (see Section 9.3.7).    -   Measure vital signs (body temperature, pulse, blood pressure,        see Section 9.3.7).    -   Collect blood samples for binding antibody testing to RBD of S        protein of SARS CoV-2 (˜6 mL blood); SARS-CoV-2 viral        neutralizing activity (˜6 mL blood); and binding antibody        testing to N protein of SARS-CoV-2 (˜6 mL blood).

Inform the subject that they have completed the main part of the trialand that the extension part of the trial will now begin (see Section9.1.4).

9.1.2 Phase 2b: Non-Immunogenicity Subjects

Following enrollment of subjects into the Immunogenicity Subset of Phase2b (n=1,200), the remaining 2,800 subjects, 18 years of age and older,will be enrolled into Phase 2b.

9.1.2.1 Clinic Visit 1: Day 1—First Trial Vaccination

Note that procedures to establish subject eligibility, recording ofdemographic information and medical history may be performed within 21days prior to trial vaccine administration, i.e., spread out over morethan 1 day. However, if all information is available and assessments andprocedures can be performed, eligibility can be established on the sameday of trial vaccine administration. All eligibility criteria must bereviewed prior to trial vaccine administration on Day 1.

Pre-Vaccination Procedures

-   -   Obtain the signed informed consent form.    -   Signed informed consent must be obtained prior to the subject        entering into the trial, and before any protocol-directed        procedures are performed (see Section 12.4).    -   By signing the informed consent form, the subject voluntarily        agrees to participate in the HERALD Trial CV-NCOV-004 and its 1        year Extension Study for a total of approximately 2 years.    -   Review inclusion/exclusion criteria (see Section 6.1 and 6.2)        and review prohibited medications listed as an exclusion        criterion (see Section 6.2).    -   Record demographic information.    -   Record medical history.    -   Record concomitant medication and vaccination, including        recurring medication for intermittent conditions, if taken        within 6 months prior to enrollment in this trial.    -   Perform a complete physical examination, including height and        weight (see Section 9.3.7). If the complete physical examination        to establish eligibility was performed within 21 days prior to        trial vaccine administration, a symptom-directed physical        examination should be performed on the day of vaccination prior        to trial vaccine administration.    -   Measure vital signs (body temperature, pulse, blood pressure,        see Section 9.3.7).    -   Perform urine pregnancy test in females of childbearing        potential.    -   Collect pre-vaccination blood sample) for binding antibody        testing to N protein of SARS-CoV-2 (˜6 mL blood).

Vaccination Procedure

-   -   Review criteria for delay or cancellation of vaccination. See        Sections 6.3 and 8.1 for an overview of the criteria leading to        delay or cancellation of vaccine administration. In case of        delay, the vaccination should take place within the allowed time        windows. The reasons for delay or cancellation should be        documented in the subject chart.    -   Administer the trial vaccine dose according to the subject's        assignment.

Post-Vaccination Procedures

-   -   Observe the subject on site for at least 30 minutes following        vaccination for safety monitoring. At the end of the observation        period:    -   Measure vital signs (body temperature, pulse, blood pressure,        see Section 9.3.7).    -   The subject may not be discharged until vital signs are within        normal range or have returned to pre-vaccination levels.    -   Record the occurrence of any AEs following trial vaccination.    -   Instructions for the subject:    -   Instruct the subject how to measure solicited AEs and how to        complete the eDiary. The subject should record solicited local        and systemic AEs occurring on the day of vaccination and the        following 7 days, and unsolicited AEs (i.e., the occurrence of        all other AEs) occurring on the day of vaccination and the        following 28 days.    -   Remind the subject to call the site immediately to report the        following:    -   If he/she experiences any concerning local or systemic reactions        or other medical event.    -   Any medically-attended visits that are not routine visits for        physical examination or vaccination, such as visits for        hospitalization, an emergency room visit, or an otherwise        unscheduled visit to or from medical personnel (medical doctor)        for any reason.    -   Experience a serious medical event, have a change in overall        health or be diagnosed with a new medical condition by a doctor.        These should be reported regardless of the perceived        relationship between the event and the trial vaccine.    -   Remind the subject to contact the site immediately if he/she has        any of the symptoms suggestive of COVID-19. In addition,        subjects will be messaged up to twice a week to provide a yes or        no response to having COVID-19 symptoms. Those who respond “yes”        will be contacted by trial staff for follow-up information (see        Section 9.2.1 and Section 9.5).    -   The subject should also be reminded to contact the site        immediately if he/she had a positive SARS-CoV-2 test performed        outside of the site, whether they were symptomatic (COVID-19        illness) or asymptomatic at the time of the test.

Note: Subjects without symptoms may have been tested for severalreasons, for example, close exposure to a known person with SARS-CoV-2infection or as part of their routine screening as a healthcareprovider.

9.1.2.2 Phone Call: Day 2 (−0/+0 day)

The purpose of this phone contact is to inquire about the subject'sgeneral well-being and to assess safety 1 day after the first trialvaccination.

-   -   During the phone call:    -   Review and record any newly reported safety data including        solicited and unsolicited AEs, or other AEs (medically-attended        AEs, SAEs).    -   Record concomitant medications and vaccinations, including        recurring medications for intermittent conditions.    -   If the subject reports any concerning local or systemic        reactions, or other AEs (e.g., medically-attended AEs, SAEs),        these should be followed-up either by a phone call(s) or by an        unscheduled site visit based on the judgment of the        investigator.    -   Instructions for the subject:    -   Remind the subject to continue recording solicited and        unsolicited AEs (i.e., the occurrence of all other AEs) in the        eDiary.    -   Remind the subject to call the site immediately to report the        following:    -   If he/she experiences any concerning local or systemic reactions        or other medical event.    -   Any medically-attended visits that are not routine visits for        physical examination or vaccination, such as visits for        hospitalization, an emergency room visit, or an otherwise        unscheduled visit to or from medical personnel (medical doctor)        for any reason.    -   Experience a serious medical event, have a change in overall        health or be diagnosed with a new medical condition by a doctor.        These should be reported regardless of the perceived        relationship between the event and the trial vaccine.    -   Remind the subject to contact the site immediately if he/she has        any of the symptoms suggestive of COVID-19. In addition,        subjects will be messaged up to twice a week to provide a yes or        no response to having COVID-19 symptoms. Those who respond “yes”        will be contacted by trial staff for follow-up information (see        Section 9.2.1 and Section 9.5).    -   The subject should also be reminded to contact the site        immediately if he/she had a positive SARS-CoV-2 test performed        outside of the site, whether they were symptomatic (COVID-19        illness) or asymptomatic at the time of the test.

9.1.2.3 Clinic Visit 2: Day 29—Second Trial Vaccination (−3/+7 days)

Pre-Vaccination Procedures

-   -   Review and record any newly reported safety data including        solicited and unsolicited AEs, or other AEs (medically-attended        AEs, SAEs).    -   Record concomitant medications and vaccinations, including        recurring medications for intermittent conditions.    -   Perform a symptom-directed physical examination (see Section        9.3.7).    -   Measure vital signs (body temperature, pulse, blood pressure,        see Section 9.3.7).    -   Perform urine pregnancy test in females of childbearing        potential.

Vaccination Procedure

-   -   Review criteria for delay or cancellation of vaccination. See        Sections 6.3 and 8.1 for an overview of the criteria leading to        delay or cancellation of vaccine administration. In case of        delay, the vaccination should take place within the allowed time        windows. The reasons for delay or cancellation should be        documented in the subject chart.    -   Administer the trial vaccine dose according to the subject's        assignment.

Post-Vaccination Procedures

-   -   Observe the subject on site for at least 30 minutes following        vaccination for safety monitoring. At the end of the observation        period:    -   Measure vital signs (body temperature, pulse, blood pressure,        see Section 9.3.7).    -   The subject may not be discharged until vital signs are within        normal range or have returned to pre-vaccination levels.    -   Record the occurrence of any AEs following trial vaccination.    -   Instructions for the subject:    -   Re-instruct the subject how to measure solicited AEs and how to        complete the eDiary. The subject should record solicited local        and systemic AEs occurring on the day of vaccination and the        following 7 days, and unsolicited AEs (i.e. the occurrence of        all other AEs) occurring on the day of vaccination and the        following 28 days.    -   Remind the subject to call the site immediately to report the        following:    -   If he/she experiences any concerning local or systemic reactions        or other medical event.    -   Any medically-attended visits that are not routine visits for        physical examination or vaccination, such as visits for        hospitalization, an emergency room visit, or an otherwise        unscheduled visit to or from medical personnel (medical doctor)        for any reason.    -   Experience a serious medical event, have a change in overall        health or be diagnosed with a new medical condition by a doctor.        These should be reported regardless of the perceived        relationship between the event and the trial vaccine.    -   Remind the subject to contact the site immediately if he/she has        any of the symptoms suggestive of COVID-19. In addition,        subjects will be messaged up to twice a week to provide a yes or        no response to having COVID-19 symptoms. Those who respond “yes”        will be contacted by trial staff for follow-up information (see        Section 9.2.1 and Section 9.5).    -   The subject should also be reminded to contact the site        immediately if he/she had a positive SARS-CoV-2 test performed        outside of the site, whether they were symptomatic (COVID-19        illness) or asymptomatic at the time of the test.

9.1.2.4 Phone Call: Day 30 (0/+0 day)

The purpose of this phone contact is to inquire about the subject'sgeneral well-being and to assess safety 1 day after the second trialvaccination.

The assessments and procedures are identical to those performed duringthe phone call on Day 2.

9.1.2.5 Clinic Visit 3: Day 43 (−3/+3 days)

-   -   Review and record any newly reported safety data including        solicited and unsolicited AEs, or other AEs (medically-attended        AEs, SAEs).    -   Record concomitant medications and vaccinations, including        recurring medications for intermittent conditions.    -   Perform a symptom-directed physical examination (see Section        9.3.7).    -   Measure vital signs (body temperature, pulse, blood pressure,        see Section 9.3.7).    -   Collect blood sample for binding antibody testing to N protein        of SARS-CoV-2 (˜6 mL blood).    -   Instructions for the subject:    -   Inform the subject that recording of solicited local and        systemic reactions in the eDiary is complete. Remind the subject        to continue recording unsolicited AEs (all AEs).    -   Remind the subject to call the site immediately to report the        following:    -   If he/she experiences any concerning medical event.    -   Any medically-attended visits that are not routine visits for        physical examination or vaccination, such as visits for        hospitalization, an emergency room visit, or an otherwise        unscheduled visit to or from medical personnel (medical doctor)        for any reason.    -   Experience a serious medical event, have a change in overall        health or be diagnosed with a new medical condition by a doctor.        These should be reported, regardless of the perceived        relationship between the event and the trial vaccine.    -   Remind the subject to contact the site immediately if he/she has        any of the symptoms suggestive of COVID-19. In addition,        subjects will be messaged up to twice a week to provide a yes or        no response to having COVID-19 symptoms. Those who respond “yes”        will be contacted by trial staff for follow-up information (see        Section 9.2.1 and Section 9.5).    -   The subject should also be reminded to contact the site        immediately if he/she had a positive SARS-CoV-2 test performed        outside of the site, whether they were symptomatic (COVID-19        illness) or asymptomatic at the time of the test.

9.1.2.6 Phone Call: Day 57 (−3/+7)

The purpose of this phone contact is to inquire about the subject'sgeneral well-being and to assess safety since site visit on Day 43.

-   -   During the phone call:    -   Review and record any newly reported safety data including        unsolicited AEs or other AEs (medically-attended AEs, SAEs).    -   Record concomitant medications and vaccinations, including        recurring medications for intermittent conditions.    -   If the subject reports any concerning local or systemic        reactions, or other AEs (e.g., medically-attended AEs, SAEs),        these should be followed-up either by a phone call(s) or by an        unscheduled site visit based on the judgment of the        investigator.    -   Instructions for the subject:    -   Inform the subject that reporting of unsolicited AEs is        complete.    -   Remind the subject to call the site immediately to report the        following:    -   If he/she experiences any concerning medical event.    -   Any medically-attended visits that are not routine visits for        physical examination or vaccination, such as visits for        hospitalization, an emergency room visit, or an otherwise        unscheduled visit to or from medical personnel (medical doctor)        for any reason.    -   Experience a serious medical event, have a change in overall        health or be diagnosed with a new medical condition by a doctor.        These should be reported regardless of the perceived        relationship between the event and the trial vaccine.    -   Remind the subject to contact the site immediately if he/she has        any of the symptoms suggestive of COVID-19. In addition,        subjects will be messaged up to twice a week to provide a yes or        no response to having COVID-19 symptoms. Those who respond “yes”        will be contacted by trial staff for follow-up information (see        Section 9.2.1 and Section 9.5).    -   The subject should also be reminded to contact the site        immediately if he/she had a positive SARS-CoV-2 test performed        outside of the site, whether they were symptomatic (COVID-19        illness) or asymptomatic at the time of the test.

9.1.2.7 Clinic Visit 4: Day 120 (−7/+7)

-   -   Review and record any newly reported AEs since the phone call on        Day 57 (medically attended AEs, SAEs).    -   Record concomitant medications and vaccinations, including        recurring medications for intermittent conditions.    -   Perform a symptom-directed physical examination (see Section        9.3.7).    -   Measure vital signs (body temperature, pulse, blood pressure,        see Section 9.3.7).    -   Instructions for the subject:    -   Remind the subject to call the site immediately to report the        following:    -   If he/she experiences any concerning medical event.    -   Any medically-attended visits that are not routine visits for        physical examination or vaccination, such as visits for        hospitalization, an emergency room visit, or an otherwise        unscheduled visit to or from medical personnel (medical doctor)        for any reason.    -   Experience a serious medical event, have a change in overall        health or be diagnosed with a new medical condition by a doctor.        These should be reported, regardless of the perceived        relationship between the event and the trial vaccine.    -   Remind the subject to contact the site immediately if he/she has        any of the symptoms suggestive of COVID-19. In addition,        subjects will be messaged up to twice a week to provide a yes or        no response to having COVID-19 symptoms. Those who respond “yes”        will be contacted by trial staff for follow-up information (see        Section 9.2.1 and Section 9.5).    -   The subject should also be reminded to contact the site        immediately if he/she had a positive SARS-CoV-2 test performed        outside of the site, whether they were symptomatic (COVID-19        illness) or asymptomatic at the time of the test.

9.1.2.8 Clinic Visit 5: Day 211 (−7/+7)

The assessments and procedures are identical to those performed duringClinic Visit 4 on Day 120, except for the below.

-   -   Collect a blood sample for binding antibody testing to N protein        of SARS CoV-2 (˜6 mL blood). 9.1.2.9 Phone Call: Day 302 (−7/+7)

The purpose of this phone contact is to inquire about the subject'sgeneral well-being and to assess safety since the site visit on Day 211.

-   -   During the phone call:    -   Review and record any newly reported AEs since the site visit on        Day 211 (SAEs).    -   Record concomitant medications and vaccinations, including        recurring medications for intermittent conditions.    -   If the subject reports by phone any concerning AEs, these should        be followed-up either by a phone call(s) or by an unscheduled        site visit based on the judgment of the investigator.    -   Instructions for the subject:    -   Remind the subject to call the site immediately to report the        following:    -   If he/she experiences any concerning medical event.    -   Any medically-attended visits that are not routine visits for        physical examination or vaccination, such as visits for        hospitalization, an emergency room visit, or an otherwise        unscheduled visit to or from medical personnel (medical doctor)        for any reason.    -   Experience a serious medical event, have a change in overall        health or be diagnosed with a new medical condition by a doctor.        These should be reported regardless of the perceived        relationship between the event and the trial vaccine.    -   Remind the subject to contact the site immediately if he/she has        any of the symptoms suggestive of COVID-19. In addition,        subjects will be messaged up to twice a week to provide a yes or        no response to having COVID-19 symptoms. Those who respond “yes”        will be contacted by trial staff for follow-up information (see        Section 9.2.1 and Section 9.5).    -   The subject should also be reminded to contact the site        immediately if he/she had a positive SARS-CoV-2 test performed        outside of the site, whether they were symptomatic (COVID-19        illness) or asymptomatic at the time of the test.

9.1.2.10 End of Trial Clinic Visit: Day 393 (−0/+21 days)

The end of trial visit will be performed on Day 393, 1 year after thelast trial vaccine administration. If possible, this visit shouldinclude subjects who prematurely discontinued vaccination during thetrial. The following assessments should be performed:

-   -   Review and record any newly reported AEs since the phone contact        on Day 302 (SAEs).    -   Record concomitant medications and vaccinations, including        recurring medications for intermittent conditions.    -   Perform a complete physical examination, including height and        weight (see Section 9.3.7).    -   Measure vital signs (body temperature, pulse, blood pressure,        see Section 9.3.7).    -   Collect a blood sample for binding antibody testing to N protein        of SARS CoV-2 (˜6 mL blood).

Inform the subject that they have completed the main part of the trialand that the extension part of the trial will now begin (see Section9.1.4).

9.1.3 Phase 3 Subjects

Approximately 32,500 subjects, 18 years of age and older, will beenrolled into Phase 3.

9.1.3.1 Clinic Visit 1: Day 1—First Trial Vaccination

Note that procedures to establish subject eligibility, recording ofdemographic information and medical history may be performed within 21days prior to trial vaccine administration, i.e., spread out over morethan 1 day. However, if all information is available and assessments andprocedures can be performed, eligibility can be established on the sameday of trial vaccine administration. All eligibility criteria must bereviewed prior to trial vaccine administration on Day 1.

Pre-Vaccination Procedures

-   -   Obtain the signed informed consent form.    -   Signed informed consent must be obtained prior to the subject        entering into the trial, and before any protocol-directed        procedures are performed (see Section 12.4).    -   By signing the informed consent form, the subject voluntarily        agrees to participate in the HERALD Trial CV-NCOV-004 and its 1        year Extension Study for a total of approximately 2 years.    -   Review inclusion/exclusion criteria (see Section 6.1 and 6.2)        and review prohibited medications listed as an exclusion        criterion (see Section 6.2).    -   Record demographic information.    -   Record medical history.    -   Record concomitant medications and vaccinations, including        recurring medications for intermittent conditions, if taken        within 6 months prior to enrollment in this trial.    -   Perform a complete physical examination, including height and        weight (see Section 9.3.7). If the complete physical examination        to establish eligibility was performed within 21 days prior to        trial vaccine administration, a symptom-directed physical        examination should be performed on the day of vaccination prior        to trial vaccine administration.    -   Measure vital signs (body temperature, pulse, blood pressure,        see Section 9.3.7).    -   Perform urine pregnancy test in females of childbearing        potential    -   Collect a pre-vaccination blood sample for binding antibody        testing to N protein of SARS-CoV-2 (˜6 mL blood).

Vaccination Procedure

-   -   Review criteria for delay or cancellation of vaccination. See        Sections 6.3 and 8.1 for an overview of the criteria leading to        delay or cancellation of vaccine administration. In case of        delay, the vaccination should take place within the allowed time        windows. The reasons for delay or cancellation should be        documented in the subject chart.    -   Administer the trial vaccine dose according to the subject's        assignment.

Post-Vaccination Procedures

-   -   Observe the subject on site for at least 30 minutes following        vaccination for safety monitoring. At the end of the observation        period:    -   Measure vital signs (body temperature, pulse, blood pressure,        see Section 9.3.7).    -   The subject may not be discharged until vital signs are within        normal range or have returned to pre-vaccination levels.    -   Record the occurrence of any new AEs following trial        vaccination.    -   Instructions for the subject:    -   Remind the subject to call the site immediately to report the        following:    -   If he/she experiences any concerning local or systemic reactions        or other medical event.    -   Any medically-attended visits that are not routine visits for        physical examination or vaccination, such as visits for        hospitalization, an emergency room visit, or an otherwise        unscheduled visit to or from medical personnel (medical doctor)        for any reason.    -   Experience a serious medical event, have a change in overall        health or be diagnosed with a new medical condition by a doctor.        These should be reported regardless of the perceived        relationship between the event and the trial vaccine.    -   Remind the subject to contact the site immediately if he/she has        any of the symptoms suggestive of COVID-19. In addition,        subjects will be messaged up to twice a week to provide a yes or        no response to having COVID-19 symptoms. Those who respond “yes”        will be contacted by trial staff for follow-up information (see        Section 9.2.1 and Section 9.5).    -   The subject should also be reminded to contact the site        immediately if he/she had a positive SARS-CoV-2 test performed        outside of the site, whether they were symptomatic (COVID-19        illness) or asymptomatic at the time of the test.

Note: Subjects without symptoms may have been tested for severalreasons, for example, close exposure to a known person with SARS-CoV-2infection or as part of their routine screening as a healthcareprovider).

9.1.3.2 Clinic Visit 2: Day 29—Second Trial Vaccination (−3/+7 days)

Pre-Vaccination Procedures

-   -   Review and record any newly collected safety data including        medically-attended AEs and SAEs.    -   Record concomitant medications and vaccinations, including        recurring medications for intermittent conditions.    -   Perform a symptom-directed physical examination (see Section        9.3.7).    -   Measure vital signs (body temperature, pulse, blood pressure,        see Section 9.3.7).    -   Perform urine pregnancy test in females of childbearing        potential.

Vaccination Procedure

-   -   Review criteria for delay or cancellation of vaccination. See        Sections 6.3 and 8.1 for an overview of the criteria leading to        delay or cancellation of vaccine administration. In case of        delay, the vaccination should take place within the allowed time        windows. The reasons for delay or cancellation should be        documented in the subject chart.    -   Administer the trial vaccine dose according to the subject's        assignment.

Post-Vaccination Procedures

-   -   Observe the subject on site for at least 30 minutes following        vaccination for safety monitoring. At the end of the observation        period:    -   Measure vital signs (body temperature, pulse, blood pressure,        see Section 9.3.7).    -   The subject may not be discharged until vital signs are within        normal range or have returned to pre-vaccination levels.    -   Record the occurrence of any new AEs following trial        vaccination.    -   Instructions for the subject:    -   Remind the subject to call the site immediately to report the        following:    -   If he/she experiences any concerning local or systemic reactions        or other medical event.    -   Any medically-attended visits that are not routine visits for        physical examination or vaccination, such as visits for        hospitalization, an emergency room visit, or an otherwise        unscheduled visit to or from medical personnel (medical doctor)        for any reason.    -   Experience a serious medical event, have a change in overall        health or be diagnosed with a new medical condition by a doctor.        These should be reported regardless of the perceived        relationship between the event and the trial vaccine.    -   Remind the subject to contact the site immediately if he/she has        any of the symptoms suggestive of COVID-19. In addition,        subjects will be messaged up to twice a week to provide a yes or        no response to having COVID-19 symptoms. Those who respond “yes”        will be contacted by trial staff for follow-up information (see        Section 9.2.1 and Section 9.5).    -   The subject should also be reminded to contact the site        immediately if he/she had a positive SARS-CoV-2 test performed        outside of the site, whether they were symptomatic (COVID-19        illness) or asymptomatic at the time of the test.

9.1.3.3 Clinic Visit 3: Day 43 (−3/+3 days)

-   -   Review and record any newly collected safety data including        medically-attended AEs and SAEs.    -   Record concomitant medications and vaccinations, including        recurring medications for intermittent conditions.    -   Perform a symptom-directed physical examination (see Section        9.3.7).    -   Measure vital signs (body temperature, pulse, blood pressure,        see Section 9.3.7).    -   Collect a blood sample for binding antibody testing to N protein        of SARS CoV-2 (˜6 mL blood).    -   Instructions for the subject:    -   Remind the subject to call the site immediately to report the        following:    -   If he/she experiences any concerning local or systemic reactions        or other medical event.    -   Any medically-attended visits that are not routine visits for        physical examination or vaccination, such as visits for        hospitalization, an emergency room visit, or an otherwise        unscheduled visit to or from medical personnel (medical doctor)        for any reason.    -   Experience a serious medical event, have a change in overall        health or be diagnosed with a new medical condition by a doctor.        These should be reported regardless of the perceived        relationship between the event and the trial vaccine.    -   Remind the subject to contact the site immediately if he/she has        any of the symptoms suggestive of COVID-19. In addition,        subjects will be messaged up to twice a week to provide a yes or        no response to having COVID-19 symptoms. Those who respond “yes”        will be contacted by trial staff for follow-up information (see        Section 9.2.1 and Section 9.5).    -   The subject should also be reminded to contact the site        immediately if he/she had a positive SARS-CoV-2 test performed        outside of the site, whether they were symptomatic (COVID-19        illness) or asymptomatic at the time of the test.

9.1.3.4 Phone Call: Day 57 (−3/+7 days) and Day 120 (−7/+7 days)

The purpose of these phone contacts is to inquire on the subject'sgeneral well-being and to assess safety since the last phone contact orsite visit.

-   -   During the phone call:    -   Review and record any newly reported AEs since the site visit or        phone call (medically attended AEs, SAEs).    -   Record concomitant medications and vaccinations, including        recurring medications for intermittent conditions.    -   If the subject reports by phone any concerning AEs, these should        be followed-up either by a phone call(s) or by an unscheduled        site visit based on the judgment of the investigator.    -   Instructions for the subject:    -   Remind the subject to call the site immediately to report the        following:    -   If he/she experiences any concerning medical event.    -   Any medically-attended visits that are not routine visits for        physical examination or vaccination, such as visits for        hospitalization, an emergency room visit, or an otherwise        unscheduled visit to or from medical personnel (medical doctor)        for any reason.    -   Experience a serious medical event, have a change in overall        health or be diagnosed with a new medical condition by a doctor.        These should be reported regardless of the perceived        relationship between the event and the trial vaccine.    -   Remind the subject to contact the site immediately if he/she has        any of the symptoms suggestive of COVID-19. In addition,        subjects will be messaged up to twice a week to provide a yes or        no response to having COVID-19 symptoms. Those who respond “yes”        will be contacted by trial staff for follow-up information (see        Section 9.2.1 and Section 9.5).    -   The subject should also be reminded to contact the site        immediately if he/she had a positive SARS-CoV-2 test performed        outside of the site, whether they were symptomatic (COVID-19        illness) or asymptomatic at the time of the test.

9.1.3.5 Clinic Visit 4: Day 211 (−7/+7 days)

The assessments and procedures are identical to those performed duringthe clinical visit on Day 43.

9.1.3.6 Phone Call: Day 302 (−7/+7 days)

The purpose of this phone contact is to inquire on the subject's generalwell-being and to assess safety since the last site visit on Day 211.

The assessments and procedures are identical to those performed duringthe phone calls on Day 57 and Day 120.

9.1.3.7 End of Trial Clinic Visit: Day 393 (−0/+21 days)

The end of trial visit will be performed on Day 393, 1 year after thelast trial vaccine administration. If possible, this visit shouldinclude subjects who prematurely discontinued vaccination during thetrial. The following assessments should be performed:

-   -   Review and record any newly reported AEs since the phone contact        on Day 302 (SAEs).    -   Record concomitant medications and vaccinations, including        recurring medications for intermittent conditions.    -   Perform a complete physical examination, including height and        weight (see Section 9.3.7).    -   Measure vital signs (body temperature, pulse, blood pressure,        see Section 9.3.7).    -   Collect a blood sample for binding antibody testing to N protein        of SARS CoV-2 (˜6 mL blood).    -   Inform the subject that they have completed the main part of the        trial and that the extension part of the trial will now begin        (see Section 9.1.4).

9.1.4 Extension Study (Up to 1 Year Duration)

-   -   General instructions for all subjects:    -   Inform the subject that the Extension Study will begin on the        last day (Day 393) of the main trial. Explain that the duration        of the trial is planned for 1 year, but may terminate early if        CVnCoV meets regulatory approval and subjects in the placebo        group are offered vaccination with CVnCoV. The trial may also        terminate early if another effective vaccine is deployed        locally.    -   Instructions for Phase 2b subjects who participated in the        Immunogenicity Subset:    -   Inform subjects that the following assessments and procedures        will be performed:    -   Return to the site every 3 months (Day 484, Day 575, Day 665,        and Day 757) for blood samples to be taken for evaluation of        long-term persistence of binding antibodies to the RBD of S        protein of SARS-CoV-2 and SARS-CoV-2 viral neutralizing        antibodies.    -   COVID-19 case detection to assess long-term efficacy.    -   Collection of AESIs and SAEs to assess long-term safety.    -   Instructions for Phase 2b Non-Immunogenicity subjects and Phase        3 subjects.    -   Inform subjects that the following assessments and procedures        will be performed:    -   Phone contact every 3 months (Day 484, Day 575, Day 665, and        Day 757) to ensure collection of AESIs and SAEs to assess        long-term safety.    -   COVID-19 case detection to assess long-term efficacy.    -   Remind the subject to call the site immediately to report the        following:    -   If he/she experiences any concerning medical event.    -   Any medically-attended visits that are not routine visits for        physical examination or vaccination, such as visits for        hospitalization, an emergency room visit, or an otherwise        unscheduled visit to or from medical personnel (medical doctor)        for any reason.    -   Experience a serious medical event, have a change in overall        health or be diagnosed with a new medical condition by a doctor.        These should be reported regardless of the perceived        relationship between the event and the trial vaccine.    -   Remind the subject to contact the site immediately if he/she has        any of the symptoms suggestive of COVID-19. In addition,        subjects will be messaged up to twice a week to provide a yes or        no response to having COVID-19 symptoms. Those who respond “yes”        will be contacted by trial staff for follow-up information.    -   The subject should also be reminded to contact the site        immediately if he/she had a positive SARS-CoV-2 test performed        outside of the site, whether they were symptomatic (COVID-19        illness) or asymptomatic at the time of the test.

9.2 Efficacy Assessments

9.2.1 COVID-19 Cases

COVID-19 case ascertainment will occur in identical manner in both thePhase 2b and Phase 3 parts of the trial. Case detection will begin withthe identification of subjects reporting at least 1 symptom from astandardized list of symptoms consistent with COVID 19 disease. Based ona phone interview with trial staff, subjects suspected of havingCOVID-19 disease will undergo testing for SARS-CoV-2 infection,consisting of a rapid antigen test performed locally by the trial staffand a molecular-based RT-PCR test performed at a designated centrallaboratory. The testing strategy is described in Section 9.5. If thesubject is confirmed to have COVID-19, subjects will be followed untilresolution of their disease, even if the initial presentation isconsidered as mild. If the subject is hospitalized, the subject'sprogress must continue to be followed by the Investigator and amedical/discharge summary must be obtained at the end of thehospitalization.

9.2.1.1 Case Detection

9.2.1.1.1 Routine Surveillance for COVID-19

During all site visits and phone calls, subjects will be reminded tocontact the site if they have any of the following symptoms*:

-   -   Fever or chills; Shortness of breath or difficulty breathing;        New loss of taste or smell; Cough; Fatigue; Muscle or body        aches; Headache; Sore throat; Congestion or runny nose; Nausea        or vomiting; Diarrhea

*FDA Development and Licensure of Vaccines to Prevent COVID-19 guidance(US Department of Health and Human Services. Food and DrugAdministration (FDA). Guidance for Industry. Development and Licensureof Vaccines to Prevent COVID 19. 2020. Available on the world wide webatfda.gov/regulatory-information/search-fda-guidance-documents/development-and-licensure-vaccines-prevent-covid-19;Accessed October 2020, incorporated herein by reference).

Subjects will also be messaged up to twice a week to provide a yes or noresponse to having COVID-19 symptoms. For both of the trialvaccinations, messaging will not begin until 4 days after vaccination toavoid confusing vaccine-associated reactions occurring during this timeperiod (e.g., fever, chills, headache, fatigue, myalgia) with potentialCOVID-19 symptoms.

Those who report symptoms either at the site visit or by phone call, orrespond “yes” to having symptoms by messaging will be contacted by trialstaff for a follow-up phone interview. The trial staff will use ascripted interview (in which he/she has been trained on) to collectinformation about the subject's medical condition, which will be used todetermine the probability of the subject having COVID-19. If the subjectis suspected of having COVID-19 illness, he/she will undergo testing forSARS-CoV-2 infection (see next section). If suspicion is low, then asubsequent phone call(s) will be performed to assess whether thesubject's illness and symptoms have progressed and if the suspicion ofCOVID-19 has reached a sufficient level to test the subject. Based onclinical judgment, phone contact may be made as frequently as daily. Allsymptomatic subjects will be provided a thermometer and oxygensaturation monitor for home use. Trial staff will instruct subjects totake their oral body temperature and oxygen saturation levels at least 3to 4 times per day, or whenever they feel symptomatic.

The testing strategy for SARS-CoV-2 infection is presented in Section9.5. Testing will consist of 2 tests: a rapid antigen test performedlocally by the trial staff and a molecular-based RT-PCR test performedat a designated central laboratory. Depending on the Investigator andhis/her facility and trial staff, nasopharyngeal swab samples fortesting will be collected either at the site or at a home visit. Thevisit to the site or home visit by trial staff will be considered an“Illness Visit” and documented as such in the eCRF.

If the subject is virologically-confirmed to have COVID-19 by a positiveRT-PCR test, subjects will be followed until resolution of theirdisease, even if the initial presentation is considered as mild. If thesubject is hospitalized, the subject's progress must continue to befollowed by the Investigator and a discharge summary must be obtained atthe end of the hospitalization. Information on clinical symptoms andsigns, their duration and severity, and treatment and outcome of theCOVID-19 episode will be documented by trial staff and recorded in theeCRF.

Upon resolution, subjects will continue to be followed in the samemanner as those who have not presented with COVID-19 (i.e. they willreturn to routine case surveillance). A second episode of COVID-19 in asubject with prior disease will not be counted as a primary efficacycase, but will be included in the exploratory objective assessing theoccurrence of second episodes of COVID-19 in vaccinated subjects.

If the subject is not virologically-confirmed by RT-PCR testing, he/shewill return to routine surveillance for COVID-19 disease as a subjectwho is naïve to SARS-CoV-2 infection (unless determined otherwise by aseropositive test to the N protein).

9.2.1.1.2 Non-Routine Surveillance for COVID-19 (Positive Test Outsideof the Site)

Subjects will be reminded to contact the site immediately if he/she hasa positive SARS CoV-2 test performed outside of the site, whether theywere symptomatic (COVID 19 illness) or asymptomatic at the time of thetest.

If the subject was symptomatic, trial staff will use the scriptedinterview to collect information about the subject's COVID-19 symptomsand medical condition. The subject should be retested as soon asfeasible to confirm the result. A nasopharyngeal swab sample should besent to the Sponsor-designated central laboratory for RT-PCR testing;the RT-PCR test result will be considered definitive as avirologically-confirmed case of COVID-19. If the subject is confirmed tohave COVID-19, subjects will be followed until resolution of theirdisease, as described above for subjects who were detected by routinesurveillance.

If the subject is not virologically-confirmed by RT-PCR testing, he/shewill return to routine surveillance for COVID-19 disease as a subjectwho is naïve to SARS-CoV-2 infection (unless determined otherwise by aseropositive test to the N protein).

9.2.1.2 Definition of Virologically-Confirmed COVID-19 Case

A virologically-confirmed case of COVID-19 is defined as a positiveSARS-CoV-2 specific RT-PCR test in a person with clinically symptomaticdisease consisting of 1 or more of the following symptoms (based on thesame screening symptoms as above):

-   -   Fever or chills; Shortness of breath or difficulty breathing;        New loss of taste or smell; Cough; Fatigue; Muscle or body        aches; Headache; Sore throat; Congestion or runny nose; Nausea        or vomiting; Diarrhea

This definition is intended to capture all seventies ofvirologically-confirmed clinically symptomatic cases of COVID-19. Assuch, COVID-19 cases classified by severity (e.g., mild or severe) willbe a subset of these cases.

9.2.1.3 COVID-19 Case Definition for Co-Primary Efficacy Analysis

For the primary analysis of efficacy, the case must meet the followingcriteria:

-   -   Must be a virologically-confirmed case of COVID-19 defined as a        positive SARS CoV 2 specific RT-PCR test in a person with        clinically symptomatic COVID-19, as defined above in Section        9.2.1.2.    -   For the primary efficacy analyses, COVID-19 cases will be        categorized as “any severity” or of “moderate to severe”        severity.    -   Symptom onset must have occurred 15 days following the second        trial vaccination.    -   The subject must not have a history of virologically-confirmed        COVID-19 illness at enrollment or have developed a case of        virologically-confirmed COVID-19 before 15 days following the        second trial vaccination {see Section 10.2.3, Efficacy Analysis        Set (EAS) for more details}.    -   The subject must have been SARS-CoV-2 naïve at baseline and Day        43 (defined as seronegative to N protein in the blood samples        collected at baseline and Day 43).

The primary efficacy cases must be confirmed by the AdjudicationCommittee.

Day 43 is 14 days post-second dose which allows the immune response toCVnCoV to mature and reach its height following the second dose. Assuch, COVID-19 case ascertainment starting the next day at 15 daysrepresents the evaluation of full VE of CVnCoV against COVID-19 disease.

9.2.1.4 Adjudication of COVID-19 Cases

An independent Committee of clinicians will be formed to adjudicateCOVID-19 cases. The Committee will be blinded to the treatmentassignment of the subject. The cases will be adjudicated by the memberswith respect to the following questions consistent with the endpoints ofthe trial.

-   -   Is the case a virologically-confirmed case of COVID-19 defined        as a positive SARS CoV-2 specific RT-PCR test in a person with        clinically symptomatic COVID-19 with 1 or more of the symptoms        listed above in Section 9.2.1.2.    -   Was the RT-PCR test performed at the CureVac designated central        laboratory?    -   Was the symptom onset of the case 15 days following the second        vaccination? Or did it occur before 15 days following the second        trial vaccination?    -   Was the subject naïve or non-naïve to SARS-CoV-2 at baseline and        Day 43? (defined as being seronegative or seropositive to the        SARS-CoV-2 N protein).    -   Was the subject 18 to 60 years of age or 61 years of age?    -   Was the subject asymptomatic? If asymptomatic, was the RT-PCR        test positive 15 days following the second vaccination or        before?    -   Was it a mild or severe case of COVID-19 based on the provided        clinical definitions?    -   Did the subject require supplemental oxygenation? What type of        oxygen support did the subject receive?    -   Was the subject hospitalized? Was the subject admitted to the        intensive care unit?    -   Did the subject die? Due to COVID-19 or other cause?

9.2.2 Asymptomatic Cases of SARS-CoV-2 Infection

There will be no active surveillance in this trial for asymptomaticSARS-CoV-2 infections. Subjects will be reminded to contact the siteimmediately if he/she had a positive SARS CoV-2 test performed outsideof the site, whether they were symptomatic (COVID 19 illness) orasymptomatic at the time of the test. Subjects without symptoms may havebeen tested for several reasons, for example, close exposure to a knownperson with SARS-CoV-2 infection or as part of their routine screeningas a healthcare provider.

If the subject was asymptomatic, trial staff will contact the subjectimmediately to collect information about the positive SARS-CoV-2 testthe subject reported for information to be collected). The subjectshould be retested as soon as feasible to confirm the result. Anasopharyngeal swab sample should be sent to the Sponsor-designatedcentral laboratory for RT-PCR testing; a positive RT-PCR test resultwill be considered definitive as a virologically-confirmed case ofSARS-CoV-2 infection.

If the subject is confirmed to have SARS-CoV-2 infection, the subjectwill be followed by trial staff for at least 2 weeks for the developmentof any COVID-19 symptoms, to ensure that this is an asymptomaticinfection. If the subject develops COVID-19, he/she will be followed-upas a COVID-19 case. If the subject is confirmed to be asymptomatic,information will be collected by the trial staff and documented on theappropriate eCRF page.

If the subject is not virologically-confirmed by RT-PCR testing, he/shewill return to routine surveillance for COVID-19 disease as a subjectwho is naïve to SARS-CoV-2 infection (unless determined otherwise by aseropositive test to the N protein).

9.3 Safety Assessments

The safety, reactogenicity, and tolerability of a 2-dose schedule ofCVnCoV will be assessed as described below.

9.3.1 Safety Assessments Specific for Subjects in Phase 2b

-   -   Reactogenicity will be assessed daily on each vaccination day        and the following 7 days by collection of solicited local AEs        (injection site pain, redness, swelling, and itching) and        systemic AEs (fever, headache, fatigue, chills, myalgia,        arthralgia, nausea/vomiting, and diarrhea) using eDiaries. In        addition, other indicators of safety will be collected (e.g.,        body temperature).    -   The eDiary will also be used as a memory aid for the subject for        the collection of unsolicited AEs on each vaccination day and        the following 28 days.

9.3.2 Safety Assessments for all Subjects in Phase 2b and Phase 3

-   -   Medically-attended AEs will be collected through 6 months after        the second trial vaccination.    -   AESIs will be collected through 1 year after the second trial        vaccination. AESIs to be monitored include pIMDs, AESIs for        SARS-CoV-2 vaccines, and non-serious intercurrent medical        conditions that may affect the immune response to vaccination.    -   SAEs will be collected through 1 year after the second trial        vaccination.    -   AEs leading to vaccine withdrawal or trial discontinuation will        be collected through 1 year after the second trial vaccination.

{If the subject does not receive their second trial vaccination, the AEfollow-up time (6 months or 1 year) will be determined based on the datescheduled for their second vaccination on Day 29}.

-   -   The eDiary will be used as a memory aid for the subject for the        collection of medically attended AEs, AESIs, and SAEs.

9.3.3 Safety Assessments for Subjects in the 1 Year Extension Study

-   -   AESIs and SAEs will be collected for up to 1 additional year in        the Extension Study.    -   The eDiary will be used as a memory aid for the subject for the        collection of AESIs and SAEs.

9.3.4 Adverse Events

Definitions of AEs/SAEs, procedures for recording, evaluating, follow-upand reporting of AEs/SAEs/pregnancy/overdose, as well as assessments ofintensity and causality of AEs.

It is important to note that COVID-19 illness and itscomplications/sequelae are consistent with the efficacy endpoints of thetrial and, as such, should not be recorded as AEs. These data will becaptured on the relevant eCRF pages for cases of COVID-19 illness thatoccur in the trial, which are expected outcomes of the trial. Therefore,COVID-19 illness and its complications/sequelae will not be reportedaccording to the standard expedited process for SAEs, even though theevent may meet the criteria for an SAE.

9.3.4.1 Solicited Adverse Events

An eDiary will be distributed to all subjects in Phase 2b for collectionof solicited local AEs (injection site pain, redness, swelling anditching) and solicited systemic AEs (fever, headache, fatigue, chills,myalgia, arthralgia, nausea/vomiting and diarrhea) on the day ofvaccination and the following 7 days. Subjects will be given athermometer to measure body temperature orally and a measuring tape todetermine the size of local injection-site reactions. Subjects will beinstructed on how to enter the solicited AEs daily for 7 days in theeDiary.

Solicited AEs will be assessed on an intensity scale of absent, mild,moderate and severe (Table A and Table B, above). By definition, alllocal solicited AEs are considered related to trial vaccination. Forsolicited systemic AEs, the Investigator will assess the relationshipbetween trial vaccine and occurrence of each AE and make an assessmentof intensity for each AE (Table B).

If concerning to the subject or of prolonged duration, solicited Grade 3AEs should be reported to the Investigator immediately. In case ofrelated Grade 3 solicited AEs reported for more than 1 day on theeDiary, the subject will be questioned to establish the total durationof the AE as exactly as possible.

9.3.4.2 Unsolicited Adverse Events and Serious Adverse Events

Unsolicited AEs occurring on the day of vaccination and the following 28days will be recorded by Phase 2b subjects for each of the 2 trialvaccinations.

For all subjects in Phase 2b and Phase 3, medically-attended AEs will becollected through 6 months after the second trial vaccination. AESIswill be collected through 1 year after the second trial vaccination (seeSection 9.3.4.3). SAEs will be collected through 1 year after the secondtrial vaccination. In the Extension Study, AESIs and SAEs will continueto be collected for an additional 1 year.

Medically-attended AEs are defined as AEs with medically-attended visitsthat are not routine visits for physical examination or vaccination,such as visits for hospitalization, an emergency room visit, or anotherwise unscheduled visit to or from medical personnel (medicaldoctor) for any reason.

The occurrence of AEs (serious and non-serious) will be assessed bynon-directive questioning of the subject at each visit. AEs volunteeredby the subject during or between visits as eDiary entries or detectedthrough observation, physical examination, laboratory test, or otherassessments during the entire trial, will be recorded in the eCRF.Subjects should be instructed to report immediately any AEs with serioussymptoms, subjective complaints or objective changes in their well-beingto the Investigator or the site personnel, regardless of the perceivedrelationship between the event and the trial vaccine.

The Investigator will assess the relationship between trial vaccine andoccurrence of each AE/SAE.

Non-serious intercurrent medical conditions that may affect the immuneresponse to vaccination will also be collected throughout the trial.

9.3.4.3 Adverse Events of Special Interest

AESIs will be collected through 1 year after the second trialvaccination in the HERALD Trial CV NCOV 004 and up to 1 additional yearin the Extension Study. The following events will be considered as AESIduring this trial:

-   -   AEs with a suspected immune-medicated etiology of potential        immune-mediated diseases (pIMDs) which are defined supra.

Celiac disease; Crohn's disease; Ulcerative colitis; Ulcerativeproctitis; Autoimmune cholangitis; Autoimmune hepatitis; Primary biliarycirrhosis; Primary sclerosing cholangitis; Addison's disease; Autoimmunethyroiditis (including Hashimoto thyroiditis; Diabetes mellitus type I;Grave's or Basedow's disease; Antisynthetase syndrome; Dermatomyositis;Juvenile chronic arthritis (including Still's disease); Mixed connectivetissue disorder; Polymyalgia rheumatic; Polymyositis; Psoriaticarthropathy; Relapsing polychondritis; Rheumatoid arthritis;Scleroderma, (e.g., including diffuse systemic form and CREST syndrome);Spondyloarthritis, (e.g., including ankylosing spondylitis, reactivearthritis (Reiter's Syndrome) and undifferentiated spondyloarthritis);Systemic lupus erythematosus; Systemic sclerosis; Acute disseminatedencephalomyelitis, (including site specific variants (e.g.,non-infectious encephalitis, encephalomyelitis, myelitis,myeloradiculomyelitis)); Cranial nerve disorders, (e.g., includingparalyses/paresis (e.g., Bell's palsy)); Guillain-Barré syndrome, (e.g.,including Miller Fisher syndrome and other variants); Immune-mediatedperipheral neuropathies, Parsonage-Turner syndrome and plexopathies,(e.g., including chronic inflammatory demyelinating polyneuropathy,multifocal motor neuropathy, and polyneuropathies associated withmonoclonal gammopathy); Multiple sclerosis; Narcolepsy; Optic neuritis;Transverse Myelitis; Alopecia areata; Autoimmune bullous skin diseases,including pemphigus, pemphigoid and dermatitis herpetiformis; Cutaneouslupus erythematosus; Erythema nodosum; Morphoea; Lichen planus;Psoriasis; Sweet's syndrome; Vitiligo; Large vessels vasculitis (e.g.,including: giant cell arteritis such as Takayasu's arteritis andtemporal arteritis); Medium sized and/or small vessels vasculitis (e.g.,including: polyarteritis nodosa, Kawasaki's disease, microscopicpolyangiitis, Wegener's granulomatosis, Churg-Strauss syndrome (allergicgranulomatous angiitis), Buerger's disease thromboangiitis obliterans,necrotizing vasculitis and anti-neutrophil cytoplasmic antibody (ANCA)positive vasculitis (type unspecified), Henoch-Schonlein purpura,Behcet's syndrome, leukocytoclastic vasculitis); Antiphospholipidsyndrome; Autoimmune hemolytic anemia; Autoimmune glomerulonephritis(including IgA nephropathy, glomerulonephritis rapidly progressive,membranous glomerulonephritis, membranoproliferative glomerulonephritis,and mesangioproliferative glomerulonephritis); Autoimmunemyocarditis/cardiomyopathy; Autoimmune thrombocytopenia; Goodpasturesyndrome; Idiopathic pulmonary fibrosis; Pernicious anemia; Raynaud'sphenomenon; Sarcoidosis; Sjögren's syndrome; Stevens-Johnson syndrome;Uveitis).

-   -   Other AEs relevant to SARS-CoV-2 vaccine development or the        target disease include: Anaphylaxis; Vasculitides; Enhanced        disease following immunization; Multisystem inflammatory        syndrome in children; Acute Respiratory Distress Syndrome;        COVID-19 disease; Acute cardiac injury; Microangiopathy; Heart        failure and cardiogenic shock; Stress cardiomyopathy; Coronary        artery disease; Arrhythmia; Myocarditis, pericarditis;        Thrombocytopenia; Deep vein thrombosis; Pulmonary embolus;        Cerebrovascular stroke; Limb ischemia; Hemorrhagic disease;        Acute kidney injury; Liver injury; Generalized convulsion;        Guillain-Barré Syndrome; Acute disseminated encephalomyelitis;        Anosmia, ageusia; Meningoencephalitis; Chilblain-like lesions;        Single organ cutaneous vasculitis; Erythema multiforme; Serious        local/systemic AR following immunization    -   Non-serious intercurrent medical conditions that may affect the        immune response to vaccination will also be collected throughout        the trial.

9.3.5 Pregnancies

Pregnancy is an exclusion criterion for enrollment in this trial, butsubjects could potentially become pregnant during their activeparticipation in this trial.

9.3.6 Safety Laboratory Assessments

A urine sample for pregnancy testing will be taken from women ofchildbearing potential on Day 1 prior to trial vaccination to establisheligibility. A urine pregnancy test will also be performed before thesecond trial vaccination on Day 29 to continue to determine eligibility.

9.3.7 Vital Signs and Physical Examination

At all trial visits for Phase 2b and Phase 3, vital signs (bodytemperature, systolic/diastolic blood pressure and pulse) will berecorded in a standardized manner after the subject has rested in thesitting position for 5 minutes.

At the first trial visit on Day 1 and end of trial visit on Day 393 forall subjects in the HERALD Trial CV NCOV-004 a complete physicalexamination will be performed, including examination of generalappearance, eyes/ears/nose/throat, head/neck/thyroid, lymph node areas,cardiovascular system, lung/chest, abdomen and genitourinary system,extremities and neurological examination, skin examination, measurementof weight and height. At all other trial visits, a symptom directedphysical examination will be performed.

9.3.8 Medical and Surgical History

All significant findings and pre-existing conditions present in asubject prior to enrollment must be reported on the relevant medicalhistory/current medical conditions screen of the eCRF.

Information should be provided on medical and surgical history andconcomitant medical conditions specifying those ongoing on Day 1.

9.3.9 Monitoring Committees

9.3.9.1 Data and Safety Monitoring Board (DSMB)

An independent DSMB will be convened to i) oversee the safety ofsubjects participating in this trial, HERALD: CV-NCOV-004; ii) to assessthe progress and conduct of the trial; Hi) to review the cumulativesafety data from the trial; iv) to perform an ongoing review of AEs ofpotential safety concern (see Section 5.5.2); and v) to makerecommendations to the Sponsor whether to continue, modify, or pause thetrial (see Section 5.5.2).

The DSMB will have regularly scheduled meetings to perform theseresponsibilities. During these meetings, the DSMB will also be informedof the safety data being generated in other ongoing clinical trials ofCVnCoV. As described in Section 5.5.2, to further ensure subject safetyon an ongoing basis, a listing of AEs of potential safety concern willbe routinely monitored by the Chair of the DSMB (or designee) at regularintervals. As described in Section 7.3.2, the DSMB may requestunblinding of an individual subject or a specific dataset at any timeduring the trial.

In addition to safety data, the DSMB will be asked to review efficacydata at the interim analyses or possibly at other time points during thetrial for a continued assessment of the risk-benefit of the trial. Aspart of the risk-benefit analysis, the DSMB will periodically monitorCOVID-19 cases for signals of VDE. The DSMB will also be asked toperform an unblinded review(s) of the incidence rate of COVID-19 casesto recommend an increase(s) in sample size, if needed.

The DSMB Charter will describe in detail the composition and objectivesof the DSMB; the responsibilities of the DSMB, CureVac, and CRO; theschedule and conduct of the DSMB meetings; and the datasets to bereviewed. The Charter will contain the statistical analysis plan (SAP)for the DSMB.

9.3.9.2 Adjudication Committee

An independent Committee of clinicians will be formed to adjudicateCOVID-19 cases for assessment of the primary endpoint. The Committeewill be blinded to the treatment assignment of the subject. The caseswill be adjudicated by the members with respect to the questionspresented in Section 9.2.1.4. The schedule of the meetings and approachto adjudication of cases will be defined in the Charter. The CommitteeChair will attend the DSMB meetings as an ad hoc member.

9.4 Immunogenicity Assessments

Because the immunogenicity results would unblind the subject's treatmentassignment, the laboratory performing the assays will keep the resultsin strict confidence. An unblinded person, named at the start of thetrial and independent of the conduct of the trial, will periodicallyreview the quality of the immunogenicity data. This person will maintainthe results in strict confidence.

9.4.1 Antibody Responses to CVnCoV Vaccination (RBD of S Protein andViral Neutralizing Antibodies)

Antibody responses to CVnCoV vaccination will only be evaluated in thePhase 2b part of the trial and only for subjects in the ImmunogenicitySubset at the time points. In the Extension Study, antibody persistencewill be evaluated every 3 months in the second year post-vaccination.

The immune response induced by vaccination with CVnCoV will be evaluatedby 2 assays:

-   -   Binding antibodies to the SARS-CoV-2 RBD of the S protein        measured in serum by immunoassay.    -   Viral neutralizing antibodies directed against SARS-CoV-2        measured in serum by a functional activity assay.

9.4.2 Antibody Responses to SARS-CoV-2 (N Protein)

Antibody responses to SARS-CoV-2 will be evaluated in all parts of thetrial and for all subjects by measuring the binding antibodies to theSARS-CoV-2 N protein (virus antigen not contained in the vaccineconstruct) at the time points specified above and will be performed byimmunoassay.

As a measure of prior infection with SARS-CoV-2, serological status tothe N protein will be used for the following:

1. To determine, retrospectively, if subjects were naïve or non-naïve toSARS-CoV-2 infection at trial entry and on Day 43.

a. For evaluation of the efficacy of a 2-dose schedule of CVnCoV innaïve subjects, subjects would have to be seronegative to the N proteinat baseline and Day 43.

b. For evaluation of the efficacy after the first dose of CVnCoV innaïve subjects, subjects would have to be seronegative to the N proteinat baseline only.

2. To determine if vaccination with a 2-dose schedule of CVnCoV canreduce infection with SARS-CoV-2 by measuring seroconversion to the Nprotein in seronegative subjects during the trial period. As describedabove in 1a, these subjects would have to be seronegative to the Nprotein at baseline and Day 43.

9.4.3 Antibody Responses to CVnCoV Vaccination in Subjects Who Develop aCase of COVID-19

For all cases of COVID-19 that occur in the trial, the antibody responseto trial vaccination will be determined in the subject's blood samplescollected on Day 1 (pre vaccination baseline), Day 43, Day 211, and Day393 of the trial. These assays will only need to be performed forsubjects in the Phase 2b part who are not in the Immunogenicity Subsetand for Phase 3 subjects. Subjects in the Phase 2b Immunogenicity Subsetwill already have these performed as part of the cohort. These resultswill be used to explore correlates of protective immunity induced byCvnCoV vaccination.

9.4.4 Cell-mediated Immunity

CMI will be evaluated in 400 subjects: 200 who receive CVnCoV and 200who receive placebo. In each CVnCoV and placebo group, 100 subjects willbe 18 to 60 years of age and 100 subjects≥61 years of age. This isintended to be carried out in one clinical site in Europe and another inLatin American country (approximately 100 CVnCoV+100 placebo subjectsparticipating at each site).

The frequency and functionality of SARS-CoV-2 RBD of S-specific T-cellresponse after antigen stimulation will be determined in PBMC incomparison to baseline. For example, ICS to investigate Th1 response andproduction of Th2 markers will be used to investigate whethervaccination induces a Th1 shift from the baseline. Further highprofiling T cell immune responses may be investigated with othertechnologies such as ELISpot or CyTOF, analysis of genomic biomarkers orany other established assays. CMI assessment will be performed on Day 1(baseline), Day 29, Day 43, Day 120 and Day 211. Note that testing onDay 120 and Day 211 will only be performed on subjects who aredetermined as T-cell responders on Day 29 and/or Day 43.

9.5 Testing for SARS-CoV-2 Infection

9.5.1 Virological Confirmation of COVID-19 Disease

During the trial, subjects clinically suspected of having COVID-19disease will undergo testing for the SARS-CoV-2 virus as describedbelow. Sample collection for the tests may be performed at the site orat a home visit by trial staff. Ideally, samples should be collectedwithin 5 days of symptom onset. The test results will be documented onthe appropriate eCRF page.

-   -   Subjects with a clinical suspicion of COVID-19 will undergo        testing for SARS-CoV-2 infection using a rapid antigen test        performed at the site with the results provided to the subject.        Nasopharyngeal swabs will be used to collect samples for the        rapid antigen test.    -   Regardless of the result of the rapid antigen test, a        nasopharyngeal swab sample collected at the same time will be        sent to a central laboratory to perform a SARS CoV 2 specific        RT-PCR test. The RT-PCR test result will be considered        definitive for SARS-CoV-2 infection. In the unlikely event that        only 1 sample can be collected from the subject, the sample        should be tested by RT-PCR at the central laboratory.    -   If the RT-PCR test is negative, but COVID-19 is still suspected        based on the subject's exposure history and clinical        presentation, another nasopharyngeal swab sample should be taken        as soon as feasible and sent to the central laboratory for        RT-PCR testing. The RT-PCR retest result will be considered        definitive for SARS CoV-2 infection.    -   Subjects who are negative for all testing will be considered        naïve to SARS-CoV-2 infection. In the unlikely case that a        subject tests positive by the rapid antigen test but negative by        RT-PCR, the subject will still be considered naïve without a        positive virological confirmation by RT-PCR (unless determined        otherwise by a seropositive test to the N protein).

9.5.2 Confirmation of a Positive Test for SARS-CoV-2 Infection PerformedOutside of the Site

See Section 9.2.1.1.2 and Section 9.2.2 for follow-up of subjects whoreport a positive test for SARS-CoV-2 infection performed outside of thesite.

For subjects (symptomatic or asymptomatic) who report a positive testfor SARS-CoV-2 infection which was performed outside of the site,regardless of the type of test, the subject should be retested as soonas feasible to confirm the result. A nasopharyngeal swab sample shouldbe sent to the central laboratory for RT-PCR testing for confirmation.The retest result at the central laboratory will be considereddefinitive.

10 Statistical Considerations

10.1 Sample Size Determination

10.1.1 Primary Efficacy Co-Objectives

This is an event-driven trial. Sample size and power considerations arebased on the co primary objectives for demonstrating efficacy of CVnCoVin the prevention of virologically confirmed cases of COVID-19 of anyseverity or COVID-19 cases of moderate or higher severity meeting theco-primary case definitions. A group sequential design with 2 interimanalyses for cases of COVID-19 of any severity demonstrating a highlevel of efficacy or reaching futility is planned using O'Brien andFleming type error spending-function (Lan et a1.1983) and the samplesize is based on the test for one single proportion (i.e. the proportionof cases in the CVnCoV group, among all cases). The group sequentialdesign is based on the any severity COVID-19 endpoint, due to the highercase number required to meet this endpoint.

To control the type one error for the 2 co-primary objectives, theoverall 2-sided alpha of 5% has been equally split between the 2co-primary objectives. With an overall 2-sided alpha of 2.5%, a total of185 COVID-19 cases of any severity (meeting the co-primary efficacy casedefinition for COVID-19 of any severity) are needed at final analysis,to have a power of 90% to demonstrate the VE is above 30% based on thelower bound of the CI for VE, when considering the VE under thealternative hypothesis is 60% (i.e. equivalently to demonstrate theproportion of cases in the CVnCoV group is below 0.4118, based on theupper bound of the CI for proportion when considering the proportionunder the alternative hypothesis is equal to 0.2857).

With an overall 2-sided alpha of 2.5%, a total of 60 moderate to severecases of COVID-19 (meeting the co-primary efficacy case definition ofmoderate or severe COVID-19) are needed at the final analysis, to have apower of 90% to demonstrate the VE is above 20% based on the lower boundof the CI for VE when considering the VE under the alternativehypothesis is 70% (i.e. equivalently to demonstrate the proportion ofcases in the CVnCoV group is below 0.4444, based on the upper bound ofthe CI for proportion when considering the proportion under thealternative hypothesis is equal to 0.2308). If 1/3 of COVID-19 cases ofany severity are moderate to severe, then 60 moderate to severe caseswill be obtained when the total number of COVID-19 cases is 180. Thereis no interim analysis planned for this endpoint.

The two interim analyses for high efficacy or futility of the co-primaryobjective of COVID 19 cases of any severity will be performed once56/111 cases have been accrued (approximately 30%/60% of cases).

Assuming an incidence rate of COVID-19 of 0.15% per month in placebosubjects, an overall non-evaluable rate of 20% (corresponding tosubjects excluded from the EAS and drop-outs) and a VE of 60%, 36,500subjects enrolled over approximately 3 months (18,250 per vaccine group)will accrue 185 COVID-19 cases of any severity at approximately 9 monthsafter the first vaccination. A lower incidence rate, a longer enrollmentduration, or a higher non evaluable rate or VE will delay theacquisition of the 185 cases and the time of final analysis. Subjectswill be randomized to receive either CVnCoV or placebo in a 1:1 ratio,stratified by country and age group (18 to 60 and 61 years of age).

10.1.2 Key Secondary Efficacy Objectives

For the key secondary efficacy objective evaluating the prevention ofvirologically confirmed severe cases of COVID-19, a lower number ofcases will be collected at the time of final analysis compared to theprimary endpoint. Based on an analysis of a large database by Verity etal. 2019, approximately 20% of COVID-19 cases can be clinically definedas severe or critical, the latter requiring intensive care.

With 37 cases of severe COVID-19 (20% of 185 cases), the trial will have88% power to detect a lower limit of the 95% CI of the VE above 10% whenassuming the VE is 70%. The power increases to 90% if the VE againstsevere cases is 75%. With complete follow up of all evaluable subjectsfor 1 year in the HERALD Trial CV-NCOV-004, it is expected that theadditional number of COVID-19 cases accrued post-second vaccinationwould permit a more robust evaluation of CVnCoV efficacy against severedisease. This analysis will be presented in the SAP.

For the next key secondary efficacy objective, assuming that 45% ofSARS-COV-2 infections are asymptomatic (Daniel et al. 2020),approximately 300 asymptomatic infections are expected after 1 completeyear of follow-up post-second vaccination for all evaluable subjects.With this number of cases, the trial will have 80% power to detect alower limit of the 95% CI of the VE above 0% when assuming the VEagainst asymptomatic infections is 28%.

10.2 Populations for Analyses

In the Safety Analysis Set (SAS), Safety Analysis Set 2 (SAS 2), and theSolicited AEs Safety Analysis Set (SASsol), subjects will be analyzed inthe group they actually received (as “treated”).

Following the “intent to treat” principle in the Efficacy sets andPer-Protocol Sets, subjects will be analyzed in the group to which theywere randomized (as “randomized”).

10.2.1 Safety Analysis Set (SAS)

The SAS will include all subjects randomized in Phase 2b or 3 whoreceived at least one dose of CVnCoV or placebo.

The SAS will be the primary population for safety endpoints collected onall subjects (i.e. medically-attended AEs, AESI, AEs leading towithdrawal or trial discontinuation and SAEs) and for efficacyobjectives assessing efficacy after the first dose.

10.2.2 Safety Analysis Sets 2 (SAS 2, SASsol)

As solicited and unsolicited AEs are collected only for Phase 2bsubjects, these analyses will then be restricted to the Phase 2bsubjects.

The SAS 2 population will include all Phase 2b subjects of the SAS andwill be used for unsolicited AEs analysis.

The SASsol population will include all Phase 2b subjects of the SAS withat least one diary collection indicating the occurrence or lack ofoccurrence of solicited AEs and will be used for solicited AEs analysis.

10.2.3 Efficacy Analysis Set (EAS)

The EAS will include all subjects randomized in Phase 2b or Phase 3 who:

-   -   Received both doses of trial vaccine according to their        randomization (2 doses of CVnCoV or 2 doses of placebo).    -   Had not developed a virologically-confirmed case of COVID-19        before trial entry (based on exclusion criteria 1) or before 15        days following the second vaccination.    -   Had not stopped the trial before 15 days following the second        vaccination.    -   Were SARS-CoV-2 naïve at baseline (based on seronegativity to N        protein in the blood sample taken at baseline).

The EAS will be the primary analysis population for all efficacyendpoints (except for the key secondary efficacy endpoint related toseroconversion and for the efficacy endpoints evaluating efficacystarting after the first dose).

10.2.4 Efficacy Analysis Set for Seroconversion (EASS)

The EASS population will include all subjects of the EAS who testedseronegative at baseline and Day 43 for the N protein of SARS-CoV-2(i.e. at all the testing time points before 15 days following the secondvaccination) and for whom at least one serological test result for Nprotein at 15 days following the second vaccination (Day 211 or 393) isavailable for analysis.

The primary analysis of the key secondary efficacy endpoint related toseroconversion to the N protein of SARS-CoV-2 (asymptomatic infections)will be performed on this population.

10.2.5 Per Protocol Efficacy Set (PPE)

The Per Protocol Efficacy set will include EAS subjects who meet alleligibility criteria at trial entry and who have no major protocoldeviations that would impact the efficacy outcomes as specified in theSAP.

The PPE will be a supportive population for efficacy endpoints (exceptfor the key secondary efficacy endpoint related to seroconversion andfor the efficacy secondary endpoint evaluating efficacy starting afterthe first dose).

10.2.6 Per Protocol Immunogenicity Set (PPI)

The PPI set will include all Phase 2b subjects who belong to theImmunogenicity Subset (IS) {i.e. ˜first 600 subjects enrolled into eachof the 2 age groups in Phase 2b (18-60 and ≥61 years of age)} and who:

-   -   Received both doses as randomized and within the windows defined        in the protocol.    -   Have no major protocol deviations expecting to impact the        immunogenicity outcomes as specified in the SAP.    -   Have not received medical treatments (such as blood products,        immunoglobulin therapy) that may interfere with one or both of        the proposed immunogenicity measurements.    -   Have at least one blood sample collected starting at 14 days        (Day 43) post-second vaccination available for analysis.

The PPI will be the primary analysis population for SARS-CoV-2 RBD of Sprotein antibody responses and SARS-CoV-2 viral neutralizing antibody.

Subjects to be excluded from the PPE/PPI will be identified and reviewedat the Blinded Data Review Meeting held before unblinding of the trial.Major protocol deviations will be listed and summarized.

Table 18 provides a summary of primary and supportive populationsplanned for analysis of each endpoint. Other analysis populations may bedefined in the SAP.

TABLE 18 Primary and Supportive Populations for the Analysis of EachEndpoint Primary Supportive Endpoints Population Population PrimaryEfficacy Endpoints EAS PPE Primary Safety Endpoints SAEs, AESI,medically-attended AEs SAS — Secondary Efficacy Endpoints: SevereCOVID-19 EAS PPE Asymptomatic infections EASS (Seroconversion to the Nprotein) COVID-19 in ≥61 years of age EAS (≥61 PPE (≥61 years of ageyears of age subjects) subjects) All SARS-CoV-2 infection (RT-PCRpositive) EAS PPE COVID-19 after first dose SAS (naïve — subjects)Secondary Immunogenicity Endpoints: SARS-CoV-2 RBD of spike (S) PPI —protein antibody responses SARS-CoV-2 viral neutralizing antibody PPI —Safety Endpoints: Solicited AEs SASsol — Unsolicited AEs SAS 2 — AEleading to vaccine withdrawal SAS — Exploratory Efficacy Endpoints:Severity of COVID-19 EAS — Supplemental oxygenation, hospitalization,EAS SAS mechanical ventilation, death COVID-19 after first dose SAS —Second episode of COVID-19 EAS — Exploratory Immunogenicity Endpoints:RBD of S-specific T-cell PPI — response after antigen stimulation byintracellular cytokine staining (ICS) to investigate Thi response andexpression of Th2 The proportion of subjects PPI — with a detectableincrease in SARS-CoV-2 RBD of S-specific T-cell response

10.3 Statistical Analyses

10.3.1 General Considerations

Five analyses are planned: 2 interim (when 56/111 cases are reached);the final (when 185 cases are reached); the 1 year follow-up (on alldata up to Day 393 visit); and the 2 year follow-up (on all data up toend of Extension Study). An SAP for the interim and final analyses willbe prepared and finalized at the latest prior to database locks. Thisdocument will provide further details regarding the definition ofanalysis variables and analysis methodology to address all trialobjectives and the handling of missing data. All analyses planned forthe final analysis will be regenerated for the 1 year follow-up and 2year follow up analyses.

10.3.2 Demographic, Medical History, and Other Baseline Characteristics

Data will be summarized with respect to demographic and baselinecharacteristics (e.g. age, gender, height, weight), medical history,baseline immune status, and all safety measurements using descriptivestatistics (quantitative data) and contingency tables (qualitative data)overall, by vaccine group, and by age group and vaccine group.

10.3.3 Trial Vaccine Administration

The administrations of CVnCoV or control will be listed and the numberof subjects actually receiving the vaccination doses will be summarizedby vaccine group.

10.3.4 Concomitant Medication and Vaccinations

Concomitant medication/vaccination after the start of the trial will belisted and summarized by Anatomical Therapeutic Chemical term, overalland by vaccine group.

10.3.5 Efficacy Analyses

10.3.5.1 Co-Primary Efficacy Endpoint Analysis

Primary Efficacy Analysis

In primary efficacy analysis, the VE, defined as the percent reductionin the frequency of any and moderate to severe COVID-19 cases (accordingto primary case definitions) in vaccinated subjects compared withsubjects who received placebo will be calculated with exact 95%* CI asfollows:

VE=1−RR=1−(ARV/ARP)=1−{p/r(1−p)}

where

ARV=attack rate in vaccinated group=nv/Nv=number of subjects reportingat least one COVID-19 episode in the CVnCoV group/total follow-up timeof evaluable subjects in the CVnCoV group (number of person-month).

ARP=attack rate in placebo group=np/Np=number of subjects reporting atleast one COVID-19 episode in the placebo group/total follow-up time ofevaluable subjects in the placebo group (number of person-month).

RR=relative risk=ARV/ARP

p=proportion of COVID-19 cases (according to primary case definition)coming from the CVnCoV group among all cases=nv/(nv+np).

r=ratio of total follow-up time of evaluable subjects in the CVnCoVgroup over total follow-up time of evaluable subjects in the placebogroup=Nv/Np.

*Level of CI may be slightly adjusted due to the sequential design (seeSection 10.3.8).

The statistical hypotheses for the co-primary efficacy endpoints are:

H0A: VE≤30% versus H1A: VE>30%

and

H0S: VE≤20% versus H1S: VE>20%

A is related to COVID-19 cases of any severity;

S is related to moderate to severe cases of COVID-19;

The trial will be successful if either the lower limit (LL) of the exact2-sided 97.5% (to be slightly adjusted to consider the sequentialdesign) CI of VE endpoint is >30% for all COVID-19 cases of any severityor if the lower limit (LL) of the exact 2-sided 97.5% CI of VE endpointis >20% for severe to moderate COVID-19 cases.

If the 2 interim analyses and the final analysis for COVID-19 cases ofany severity are performed after 56/111 and 185 cases have beenreported, respectively, the 1-sided α-risk to consider at the time offinal analysis according to O'Brien Fleming type error spending functionwill be 0.01209 and efficacy will be demonstrated at the final analysisif 60 cases or less over 185 are in the CVnCoV group (observedVE≥52.0%); and 0.025 for the final analysis for cases of moderate tosevere COVID-19 and efficacy will be demonstrated if 17 cases or lessover 60 are in the CVnCoV group (observed VE≥60.5%). To note, the rulein terms of split of cases to demonstrate efficacy can slightly differif r≠1 (total follow-up time different in both groups).

Sensitivity Analysis

As a key sensitivity analysis, the time to first-occurrence ofvirologically-confirmed COVID 19 cases (according to primary casedefinitions) will be analyzed.

The Kaplan-Meier curves will display the estimated probabilities of notdeveloping COVID 19 and log-rank test will be performed.

The time to first-occurrence of virologically-confirmed COVID-19 (dateof symptoms onset) will start 15 days following the second vaccination.

Subjects who do not develop COVID-19 will be censored at the date oftrial termination or cut-off date for analysis whichever comes first.

An additional sensitivity analysis may include a Cox proportionalhazards regression model adjusted for relevant baseline covariatesspecified in the SAP.

More details on the analysis methods will be described in the SAP.

10.3.5.2 Secondary Efficacy Endpoints Analyses

Statistical testing of the 2 key secondary efficacy endpoints will beperformed according to the conditional hierarchical testing procedureusing the order defined in the objective/endpoints sections.Consequently:

-   -   Efficacy of CVnCoV in regard to severe cases will be        demonstrated only if there is successful demonstration of the        primary efficacy objective.    -   Efficacy of CVnCoV in regard to asymptomatic infection will be        demonstrated only if there is successful demonstration of the        primary efficacy objective and secondary objective on severe        cases.

Otherwise, these endpoints will be analyzed as exploratory endpointswithout success criteria testing.

To assess the efficacy in the prevention of severe disease andasymptomatic infections, similar analyses to those performed on theprimary efficacy endpoint will be performed. The efficacy will bedemonstrated if the LL of the exact 2-sided 95% CI of VE is above 10%for severe disease and above 0% for asymptomatic infections.

Other secondary efficacy endpoints will be analyzed similarly to theprimary efficacy endpoint but no formal testing will be performed forthose endpoints. For efficacy after the first dose, the time tofirst-occurrence of virologically-confirmed COVID-19 (date of symptomonset) will start after the first vaccination. The BoD will be analyzedusing 2 different scoring systems. Both BoD scoring systems place moreweight on efficacy against severe COVID-19 disease or severe disease asreflected by hospitalization or death. In addition, VE and associated CIwill be calculated for each of the BoD categories.

10.3.5.3 Exploratory Efficacy Endpoints Analyses

The proportions of mild and severe COVID-19 cases (according to primarycase definition) among all cases will be summarized by group.

Description of frequencies and percentages will be provided by group forsubjects who:

-   -   Need supplemental oxygenation due to COVID-19.    -   Need mechanical ventilation due to COVID-19.    -   Are hospitalized due to COVID-19.    -   Died due to COVID-19.    -   Died due to any cause.

This will be done for events occurring 15 days following the secondtrial vaccination (full VE) and then for events occurring at any timeafter the first trial vaccination.

The VE in the prevention of first episodes of virologically-confirmedcases of COVID-19 of any severity will be reassessed on all subjectswhatever their serological status at baseline for cases occurring 15days following the second trial vaccination and then for all casesoccurring after the first dose.

Finally, the number and percentage of subjects who developed a secondepisode of COVID-19 will be displayed by group.

10.3.6 Secondary and Exploratory Immunogenicity Analysis

No formal hypothesis on immunogenicity will be tested. Descriptivestatistics for the immunogenicity endpoints will be provided for eachvaccine group and overall, and by vaccine group and age groups. Datawill be presented after each vaccine dose.

The following analyses will be performed for antibody levels to theSARS-CoV-2 RBD of S protein and for neutralizing antibodies overall andseparately in subjects seronegative at baseline and in subjectsseropositive at baseline:

-   -   Geometric mean titers (GMTs) will be summarized with their 95%        CI at each blood sampling time point.    -   The Fold Change (FC) from baseline will be computed for each        subject and Geometric mean of FC (GMFC) will be displayed with        their 95% CI at each blood sampling time point after baseline.

Non detectable antibodies will be arbitrary replaced by half of thedetection cut-off for GMT and GMFC computations purpose.

For each readout, the number and percentage of subjects SARS CoV-2seronegative at baseline for who a seroconversion is observed will besummarized and presented at each blood sampling time point afterbaseline with exact 95% CI. Seroconversion is defined as detectableantibodies in the serum.

Percentages of subjects seroconverting for SARS CoV-2 RBD of S proteinantibodies and SARS CoV-2 neutralizing antibodies will be summarized.The frequency of immune cell populations induced by the vaccine will besummarized.

Further characterization of the T cell immune response may be done withother technologies like ELISpot, CyTOF and/or analysis of genomicbiomarkers.

Additional immunogenicity analyses including graphs will be described inthe SAP as applicable.

10.3.7 Safety Analysis

No formal statistical testing of safety data is planned.

The descriptive safety analyses will be performed overall, by vaccinegroup and by age group and vaccine group.

The following analyses will be done overall and separately in subjectsseronegative at baseline and in subjects seropositive at baseline forSARS-CoV-2 N protein antibody levels:

Solicited AEs: The frequencies and percentages of subjects experiencingeach solicited local and systemic AE within 7 days after eachvaccination will be presented by intensity and overall. For subjectswith more than 1 episode of the same AE within 7 days after avaccination, the maximum intensity will be used for tabulations. Similartabulations will be performed for solicited systemic AEs by relationshipto trial vaccination. Solicited local AEs will be by definitionconsidered as related to the trial vaccine. Time to onset (in days) andduration (in days) will also be summarized for each solicited local andsystemic AEs. Summary tables showing the occurrence of at least onelocal or systemic solicited AE within 7 days after each vaccination willalso be presented.

Unsolicited AEs: Unsolicited AEs including SAEs and AESIs will be codedusing the Medical Dictionary for Regulatory Activities (MedDRA) bySystem Organ Class (SOC) and Preferred Term (PT).

The frequency and percentage of subjects reporting each unsolicited AEwithin the 28 days after each vaccination and overall will be tabulatedat the SOC and PT levels.

Similar tables will be provided for: related unsolicited AEs, Grade 3 orhigher unsolicited AEs, medically-attended AEs that occur within 6months after the second trial vaccination, SAEs, related SAEs, AESIs,related AESIs, AEs leading to withdrawal or trial discontinuation andSAEs resulting in death through 1 year after the second trialvaccination. When an AE occurs more than once for a subject within the28 days post 1 vaccination, the maximal severity and strongestrelationship to the vaccine group will be counted.

Only AE post first vaccination will be considered in the summary tables.AE starting prior to the first vaccination will be recorded as medicalhistory.

Data listings of fatal and SAEs will be provided by subject.

Vital signs will be summarized by descriptive statistics at each visit,including change from baseline, and a listing will be provided.

10.3.8 Interim Analysis

Two interim analyses will be performed for this trial by an unblindedindependent statistician and reviewed by the DSMB when 56/111 cases ofCOVID-19 of any severity (meeting the co-primary efficacy casedefinition) are observed. This analysis will aim to assess early highefficacy or futility on the primary efficacy endpoint and will be doneon the EAS population only. The safety data that is available at thistime point will also be described.

For the analysis of early demonstration of high efficacy or futility,cumulative O'Brien Fleming type error spending function (Lan et a1.1983)is used to provide statistical stopping rules for high efficacy(α-boundaries) and futility (β-boundaries) for the interim analysis,based on the information accumulated until that specific interim stage.

At the interim stage, if the p-value for the test of the primaryobjective is lower than the α boundary, a high level of efficacy forCVnCoV will be declared. Conversely, demonstration of futility willoccur if the p-value is higher than the β-boundary.

The interim analyses are planned to occur when 56/11 cases of COVID-19of any severity have been observed. Table 19 below shows the boundariesfor demonstrating high efficacy or futility, calculated on a 1 sidedp-value scale using the cumulative error spending function.

TABLE 19 Two Stage Group Sequential Design with Interim Analyses at 56and 111 Cases and Final Analysis at 185 Cases Interim Interim FinalAnalysis 1 Analysis 2 Analysis Number 56 111 185 of Cases Efficacy0.00001 0.00126 0.01209 α-Boundary on p-value scale (1-sided) Futility0.73596 0.15716 NA β-Boundary on p-value scale (1-sided) EfficacySuccess Success if Success if success if ≤7 cases ≤29 cases ≤60 casescriteria* in CVnCoV in CVnCoV in CVnCoV group over 56 group over 111group over cases cases 185 cases (observed VE (observed VE (observed VE≥85.7%) ≥64.6%) ≥52.0%) Futility* Futility if ≥26 Futility if ≥41 NAcases in cases CVnCoV in CVnCoV group over group over 111 56 cases cases(observed (observed VE ≤13.3%) VE ≤41.4%) *Rules in terms of split ofcases to demonstrate efficacy/futility can slightly differ if the totalnumber of evaluable subjects is unequal in both groups (r ≠ 1).

If the interim analysis is performed exactly after 56/111 cases havebeen reported, a 1 sided p-value lower than 0.00001/0.00126 (i.e. lowerlimit of the 2-sided 99.99%/99.99% CI>30%) will lead to the conclusionof high efficacy, while a 1-sided p-value higher than 0.73596/0.15716will result in the demonstration of futility. Otherwise, the finalanalysis will be performed at 185 cases. Similarly, if the number ofevaluable subjects is equal in both groups, it means that the trial willconclude early high efficacy if 7/29 cases or less over 56/111 arecoming from the CVnCoV group, while futility of the trial will bedemonstrated if 26/41 cases or more are coming from the CVnCoV group.

Of note, the actual boundaries used for decision making would depend onthe exact number of cases occurring and reported at each analysis(interim and final).

The boundaries will be applied in a nonbinding way as there are manyother factors that would be part of the decision-making process.

10.3.9 Missing Data and Discontinuation

Analysis of vaccination variables will be done on a valid case basis,i.e., for missing observations, no imputation for missing data, such aslast observation carried forward, will be applied.

For SARS-CoV-2 RBD of S protein antibodies, concentration values markedas below the lower limit of quantification (LLOQ) will be set to0.5*LLOQ.

No imputation of missing values will be done for any analysis (exceptthe imputation for missing partial dates of AEs and concomitantmedication as specified in the SAP).

Currently no replacement of drop-out subjects is foreseen.

Example 14: Vaccination of Rats with mRNA Encoding SARS-CoV-2 AntigenS_Stab Formulated in LNPs

The present example shows that SARS-CoV-2 S mRNA vaccines with mRNAcomprising alternative forms of the 3′ end (A64-N5-C30-hSL-N5 orhSL-A100) and UTR combinations (i-3 (−/muag) or a-1 (HSD17B4/PSMB3))induce strong humoral as well as cellular immune response in rats. mRNAencoding SARS-CoV-2 S_stab comprising hSL-A100 and the UTR combinationa-1 (HSD17B4/PSMB3) shows stronger and very early induction of immuneresponses, demonstrated by a stronger induction of binding andneutralizing antibodies even after one first vaccination.

Preparation of LNP Formulated mRNA Vaccine:

SARS-CoV-2 S mRNA constructs are prepared as described in Example 1 (RNAin vitro transcription). HPLC purified mRNA was formulated with LNPsaccording to Example 1.4 prior to use in in vivo vaccinationexperiments.

Immunization:

Rats were injected intramuscularly (i.m.) with mRNA vaccine compositionsand doses as indicated in Table 18. As a negative control, one group ofrats was vaccinated with buffer (group A). All animals were vaccinatedon day 0 and day 21. Blood samples were collected on day 14, day 21(post prime) and 42 (post boost) for the determination of antibodytiters.

TABLE 20 Vaccination regimen (Example 14): 5′-UTR/ 3′-UTR; SEQ SEQ mRNACDS UTR ID NO: ID NO: Group Vaccine composition ID opt. Design 3′-endProtein RNA Dose buffer — — — — — A mRNA encoding S_stab R9515 opt1—/muag; A64-N5- 10 163 0.5 μg formulated in LNPs C30-hSL-N5 B mRNAencoding S_stab R9515 opt1 —/muag; A64-N5- 10 163   2 μg formulated inLNPs C30-hSL-N5 C mRNA encoding S_stab R9515 opt1 —/muag; A64-N5- 10 163  8 μg formulated in LNPs C30-hSL-N5 D mRNA encoding S_stab R9515 opt1—/muag; A64-N5- 10 163  20 μg formulated in LNPs C30-hSL-N5 E mRNAencoding S_stab R9515 opt1 —/muag; A64-N5- 10 163  40 μg formulated inLNPs C30-hSL-N5 F mRNA encoding S_stab R9709 opt1 HSD17B4/ hSL-A100 10149 0.5 μg formulated in LNPs PSMB3 G mRNA encoding S_stab R9709 opt1HSD17B4/ hSL-A100 10 149   2 μg formulated in LNPs PSMB3 H mRNA encodingS_stab R9709 opt1 HSD17B4/ hSL-A100 10 149   8 μg formulated in LNPsPSMB3 I mRNA encoding S_stab R9709 opt1 HSD17B4/ hSL-A100 10 149  20 μgformulated in LNPs PSMB3 J mRNA encoding S_stab R9709 opt1 HSD17B4/hSL-A100 10 149  40 μg formulated in LNPs PSMB3

Determination of IgG1 and IgG2 Antibody Titers Using ELISA:

ELISA was performed as described before in Example 12.

Determination of Virus Neutralizing Antibody Titers (VNT)

Virus neutralizing antibody titers (VNT) of rat serum samples wereanalyzed as previously described in Example 6 with mouse serum.

Results:

As shown in FIG. 16A the vaccination with mRNA full length S stabilizedprotein comprising the non-coding region with 3′ end hSL-A100 and theUTR combination a-1 (HSD17B4/PSMB3) formulated in LNPs (R9709) inducedin rats robust and dose dependent levels of binding antibody titers(shown by IgG1 and IgG2a endpoint titers) at day 14 and day 21 alreadyafter one first vaccination using doses of 0.5 μg, 2 μg, 20 μg, and 40μg. The vaccination with mRNA full length S stabilized proteincomprising the non-coding region with 3′ end A64-N5-C30-hSL-N5 and theUTR combination i-3 (−/muag) formulated in LNPs (R9515, CVnCov) inducedin rats dose dependent levels of binding antibody titers (shown by IgG1and IgG2a endpoint titers) at day 14 and day 21 already after one firstvaccination using the higher doses (Ng, 20 μg, and 40 μg).

As shown in FIG. 16B vaccination with mRNA comprising the non-codingregion with 3′ end hSL-A100 and the UTR combination a-1 (HSD17B4/PSMB3)encoding full length S stabilized protein formulated in LNPs (R9709)induced in rats dose dependent and very high levels of VNT, alreadyafter 14 days after one first vaccination with a dose of at least 2 μg.

As shown in FIG. 16C vaccination with both mRNA vaccine formats encodingfull length S stabilized protein formulated in LNPs induce strong VNTsin a dose dependent manner. The induction of VNTs with mRNA encodingSARS-CoV-2 S_stab comprising hSL-A100 and the UTR combination a-1(HSD17B4/PSMB3) shows a stronger and very robust induction of very highneutralizing antibody titers even at a dose of only 2 μg, when comparedwith mRNA encoding SARS-CoV-2 S_stab comprising A64-N5-C30-hSL-N5 andthe UTR combination i-3 (−/muag). The titer of neutralizing antibodiesraised by the vaccine composition comprising mRNA R9709 could be furthernotably increased by the second vaccination.

The strength of vaccine composition comprising R9709 may support animmunization protocol for the treatment or prophylaxis of a subjectagainst coronavirus, preferably SARS-CoV-2 coronavirus comprising onlyone single dose of the composition or the vaccine.

Example 15: Vaccination of NHP with mRNA Encoding SARS-CoV-2 AntigenS_Stab Formulated in LNPs and Challenge

The protective efficacy of mRNA encoding S_stab formulated in LNPs(CVnCoV) was addressed in a rhesus macaque SARS-CoV-2 challenge model.Non-human primates develop mild clinical disease with high levels ofviral replication in both the upper and lower respiratory tract andpathological changes indicative of viral pneumonia upon infection withSARS-CoV-2 (Munoz-Fontela et al., 2020). Results presented that CVnCoVhad protective impact against challenge with 5×10⁶ PFU via the intranasal (IN) and intra tracheal (IT) routes in an NHP in vivo model ofCOVID-19. Protective endpoints include significantly reduced virus load,in addition to protection against lung pathology.

Preparation of LNP Formulated mRNA Vaccine:

SARS-CoV-2 S mRNA construct was prepared as described in Example 1 (RNAin vitro transcription). HPLC purified mRNA was formulated with LNPsaccording to Example 1.4 prior to use in in vivo vaccinationexperiments.

Immunization and Challenge:

Eighteen rhesus macaques (Macaca mulatta), of Indian origin were dividedinto three groups of six, each comprising three males and three females(with a weight of >4.5 kg and an age of 3-6 years). Animals werevaccinated twice with either 0.5 μg or 8 μg LNP-formulated mRNA encodingSARS-CoV-2 antigen S_stab (SARS-CoV-2 S-2P (CVnCoV)) or remainedunvaccinated prior to challenge with wild type SARS-CoV-2 four weeksafter the second vaccination (see FIG. 17A). The animals were injectedintramuscularly (i.m.) in the bicep muscle of the upper arm with mRNAvaccine compositions, in a volume of 0.5 ml and doses as indicated inTable 21. As negative control, one group of NHPs was nottreated/unvaccinated before challenge (group A). Blood samples werecollected on day 0, 14, 28 (post first vaccination), on day 42 and 56(post second vaccination), and on day 1, 3, 5, 7 after challenge for thedetermination of antibody titers. The animals were intranasallychallenged on day 56 with a dose of 5.0×10⁶ PFU SARS-CoV-2 by applying 2ml of virus preparation to the pre-carinal section of the trachea usinga bronchoscope followed by 1 ml applied intranasally (0.5 ml/nostril).Two animals of each group were followed for 6, 7 or 8 days postchallenge (p.c.) and euthanised on day 62, 63 or 63 of the experiment.

TABLE 21 Vaccination regimen (Example 15): SEQ SEQ mRNA CDS ID NO: IDNO: Group Vaccine composition ID dose vaccination opt. Protein RNA AmRNA encoding S_stab R9515 0.5 μg d0, d28 opt1 10 163 formulated in LNPs(CVnCoV) B mRNA encoding S_stab R9515   8 μg d0, d28 opt1 10 163formulated in LNPs (CVnCoV) C Unvaccinated

IgG ELISA

A full-length trimeric and stabilised version of the SARS-CoV-2 Spikeprotein was supplied by Lake Pharma (#46328). Recombinant SARS-CoV-2Receptor-Binding-Domain (319-541) Myc-His was developed and kindlyprovided by MassBiologics. Recombinant SARS-CoV-2 Spike- andRBD-specific IgG responses were determined by ELISA. High-binding96-well plates (Nunc Maxisorp, 442404) were coated with 50 μl per wellof 2 μg/ml Spike trimer or Spike RBD in 1×PBS (Gibco) and incubatedovernight at 4° C. The ELISA plates were washed and blocked with 5%Foetal Bovine Serum (FBS, Sigma, F9665) in 1×PBS/0.1% Tween 20 for 1hour at room temperature. Serum collected from animals after vaccinationhad a starting dilution of 1/50 followed by 8, two-fold serialdilutions. Post-challenge samples were inactivated in 0.5% triton andhad a starting dilution of 1/100 followed by 8, three-fold serialdilutions. Serial dilutions were performed in 10% FBS in 1×PBS/0.1%Tween 20. After washing the plates, 50 μl/well of each serum dilutionwas added to the antigen-coated plate in duplicate and incubated for 2hours at room temperature. Following washing, anti-monkey IgG conjugatedto HRP (Invitrogen, PA1-84631) was diluted (1:10,000) in 10% FBS in1×PBS/0.1% Tween 20 and 100 μl/well was added to the plate. Plates werethen incubated for 1 hour at room temperature. After washing, 1 mg/mlO-Phenylenediamine dihydrochloride solution (Sigma P9187) was preparedand 100 μl per well were added. The development was stopped with 50 μlper well 1M Hydrochloric acid (Fisher Chemical, J/4320/15) and theabsorbance at 490 nm was read on a Molecular Devices versamax platereader using Softmax (version 7.0). All test sample dose response curveswere fitted to a 4PL model in Softmax Pro (version 7.0) and the endpointtitre at an OD of 0.5 (defined as reciprocal of the serum dilutionrequired to give an absorbance response of 0.5) was interpolated fromeach curve. Where results were below the limit of detection, they wereassigned a value of 25 for the post immunisation samples and 50 for thepost challenge samples. For low samples where the absorbance neverreached a value of 0.5, the titre was estimated from the extrapolatedportion of the curve. The cut-off was set as the average titre of serumcollected from naïve animals (day 0)+1 Standard Deviation. The cut offwas calculated separately for each antigen.

SARS-CoV-2 Focus Reduction Neutralisation Test

Virus neutralising titres were measured in heat-inactivated serumsamples (56° C. for 30 min). SARS-CoV-2 (Victoria/01/2020, DohertyInstitute) at a concentration to give 100 to 250 foci per well in thevirus only control wells was mixed 50:50 in 1% FCS MEM with 1×antibiotic/antimycotic (Gibco, 15240-062) with serum doubling dilutionsfrom 1:20 to 1:640 (or higher dependent on antibody levels) in a 96-wellV-bottomed plate. The plate was incubated at 37° C. in a humidified boxfor 1 hour to allow antibodies in the serum sample to bind to the virus.One hundred microlitres of the serum/virus mixture was then transferredto virus susceptible Vero/E6 monolayers in 96-well plates and incubatedfor a further 1 hour at 37° C. in a sealed humidified box. Afteradsorption, the virus/antibody mixture was removed and 100 μl of 1% w/vCMC in complete media overlay was added. The box was resealed andincubated at 37° C. for 24 hours prior to fixing with 100 μl of 20%formalin/PBS solution and fumigation of the plate overnight prior toimmunostaining. Following washing with water using an ELISA washer(BioTek 405 TSUS), residual endogenous peroxidase activity was removedby the addition of 0.3% hydrogen peroxide for 20 min. Plates were thenincubated for 1 h with primary/detection SARS-CoV-2 anti-RBD rabbitpolyclonal antibody (SinoBiologicals; 40592-T62) diluted 1:2,000 in PBS.After washing, plates were incubated for 1 h with secondary anti-rabbitHRP-conjugate antibody (Invitrogen; G-21234) diluted 1:4,000 in PBS.After washing, foci were visualised using TrueBlue™ Peroxidase Substrate(KPL seracare; 5510-0030) after which plates were washed with water anddried. Foci were counted using an ImmunoSpot S6 Ultra-V analyser (CTL)and BioSpot software (7.0.28.4 Professional; CTL) and the resultsanalysed in SoftMax Pro (Molecular Devices; v7.0.3 GxP). Briefly, thecount data was expressed as percentage of VOC for each serum dilution,i.e. percentage foci reduction and plotted on a 4-Parameter logistic(4PL) curve. The virus neutralisation titre (VNT) is reported as serumdilution that neutralised 50% of the virus foci.

Alternatively virus neutralizing titres were measured as previouslydescribed in Example 9 with a SARS-CoV-2 virus featuring the mutationD614G.

ELISpot

Peripheral Blood Mononuclear Cells (PBMCs) were isolated from wholeheparinised blood by density gradient centrifugation using Ficoll-PaquePlus (GE Healthcare, USA). An IFN-7 ELISpot assay was used to estimatethe frequency and IFN-γ production capacity of SARS-CoV-2-specific Tcells in PBMCs using a human/simian IFN-7 kit (MabTech, Nacka, Sweden).The cells were assayed at 2×10⁵ cells per well. Cells were stimulatedovernight with SARS-CoV-2 peptide pools and ‘megapools’ of the spikeprotein (Mimotopes, Australia). Peptide sequence was based on GenBank:MN908947.3. Ten peptide pools were used, comprising of 15 mer peptides,overlapping by 11 amino acids. The three megapools were made up as such:Megapool 1 (MP1) comprised peptide pools 1-3, Megapool 2 (MP2) comprisedpeptide pools 4-6 and Megapool 3 (MP3) comprised of peptide pools 7-10.All peptides were used at a final concentration of 1.7 μg/ml perpeptide. Phorbol 12-myristate (Sigma-Aldrich Dorset, UK) (100 ng/ml) andionomycin (CN Biosciences, Nottingham, UK) (1 mg/ml) were used as apositive control. Results were calculated to report as spot formingunits (SFU) per million cells. All SARS-CoV-2 peptides and megapoolswere assayed in duplicate and media only wells subtracted to give theantigen-specific SFU. ELISpot plates were analysed using the CTL scannerand software (CTL, Germany) and further analysis carried out usingGraphPad Prism (version 8.0.1) (GraphPad Software, USA).

Bronchioalveolar Lavage (BAL)

In-life BAL washes were performed using 6 ml or 10 ml PBS using abronchioscope inserted to the right side of the lung above the secondbifurcation. BAL washes performed post-mortem were conducted on theright lung lobes, after ligation of the left primary bronchus using 20ml PBS.

Quantitative Polymerase Chain Reaction

RNA was isolated from nasal swab, throat swabs, EDTA treated wholeblood, BAL and tissue samples (spleen, kidney, liver, colon, duodenum,tonsil, trachea and lung). Tissue samples in RNAprotect (Qiagen), werehomogenised in a Precellys 24 homogeniser with CK28 Hard tissuehomogenizing 2.0 ml tubes (Bertin) and 1 ml of RLT buffer (Qiagen)supplemented with 1% (v/v) Beta-mercaptoethanol. Tissue homogenate waspassed through a QIAshredder homogenizer (Qiagen) and a volume thatequated to 17.5 mg of tissue was extracted using the BioSprint™96One-For-All vet kit (Qiagen) and Kingfisher Flex platform as permanufacturer's instructions. Non-tissue samples were inactivated byplacing samples into AVL buffer (Qiagen) and adding 100% ethanol.Extraction of these samples was performed using the BioSprint™96One-For-All vet kit (Qiagen) and Kingfisher Flex platform as permanufacturer's instructions.

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)was performed using TaqPath™ 1-Step RT-qPCR Master Mix, CG (AppliedBiosystems™), 2019-nCoV CDC RUO Kit (Integrated DNA Technologies) andQuantStudio™ 7 Flex Real-Time PCR System (Applied Biosystems™). PCRamplicons were quantified against in vitro transcribed RNA N genefragment standard. Positive samples detected below the lower limit ofquantification (LLOQ) of 10 copies/μl were assigned the value of 5copies/μl, undetected samples were assigned the value of 2.3 copies/μl,equivalent to the assays LLOD. For nasal swab, throat swab, BAL andblood samples extracted samples this equates to an LLOQ of 1.29×10⁴copies/ml and LLOD of 2.96×10³ copies/ml. For tissue samples thisequates to an LLOQ of 5.71×10⁴ copies/g and LLOD of 1.31×10⁴ copies/g.

Subgenomic RT-qPCR was performed on the QuantStudio™ 7 Flex Real-TimePCR System using TaqMan™ Fast Virus 1-Step Master Mix (Thermo FisherScientific) with forward primer, probe and reverse primer at a finalconcentration of 250 nM, 125 nM and 500 nM respectively. Sequences ofthe sgE primers and probe were: 2019-nCoV_sgE-forward, 5′CGATCTCTTGTAGATCTGTTCTC 3′ (SEQ ID NO: 22729); 2019-nCoV_sgE-reverse, 5′ATATTGCAGCAGTACGCACACA 3′ (SEQ ID NO: 22730); 2019-nCoV_sgE-probe, 5′FAM-ACACTAGCCATCCTTACTGCGCTTCG-BHQ1 3′ (SEQ ID NO: 22731). Cyclingconditions were 50° C. for 10 minutes, 95° C. for 2 minutes, followed by45 cycles of 95° C. for 10 seconds and 60° C. for 30 seconds. RT-qPCRamplicons were quantified against an in vitro transcribed RNA standardof the full-length SARS-CoV-2 E ORF (accession number NC_045512.2)preceded by the UTR leader sequence and putative E gene transcriptionregulatory sequence. Positive samples detected below the lower limit ofquantification (LLOQ) were assigned the value of 5 copies/μl, whilstundetected samples were assigned the value of ≤0.9 copies/μl, equivalentto the assays lower limit of detection (LLOD). For nasal swab, throatswab, BAL and blood samples extracted samples this equates to an LLOQ of1.29×10⁴ copies/ml and LLOD of 1.16×10³ copies/ml. For tissue samplesthis equates to an LLOQ of 5.71×10⁴ copies/g and LLOD of 5.14×10³copies/g.

Histopathology

Tissue samples from left cranial and caudal lung lobes, trachea, larynx,mediastinal lymph node, tonsil, heart, thymus, pancreas, spleen, liver,kidney, duodenum, colon, brain, vaccinating site (skin includingsubcutis and underlying muscle) and draining lymph node (left and right)were fixed in 10% neutral-buffered formalin and embedded into paraffinwax. 4 μm thick sections were cut and stained with haematoxylin andeosin (HE). Tissue slides were scanned and examined independently by twoveterinary pathologists blinded to the treatment and group details.

For the lung, three sections from each left lung lobe were sampled fromdifferent locations: proximal, medial and distal to the primary lobarbronchus. A scoring system (Salguero et al., 2020) was used to evaluateobjectively the histopathological lesions observed in the lung tissuesections. The scores for each histopathological parameter werecalculated as the average of the scores observed in the six lung tissuesections evaluated per animal.

Additionally, RNAscope in-situ hybridisation (ISH) technique was used toidentify the SARS-CoV-2 virus in both lung lobes. Briefly, tissues werepre-treated with hydrogen peroxide for 10 mins (RT), target retrievalfor 15 mins (98-101° C.) and protease plus for 30 mins (40° C.) (allAdvanced Cell Diagnostics). A V-nCoV2019-S probe (Advanced CellDiagnostics) targeting the S-protein gene was incubated on the tissuesfor 2 hours at 40° C. Amplification of the signal was carried outfollowing the RNAscope protocol (RNAscope 2.5 HD Detection Reagent—Red)using the RNAscope 2.5 HD red kit (Advanced Cell Diagnostics).Appropriate controls were included in each ISH run. Digital imageanalysis was carried out with Nikon NIS-Ar software in order tocalculate the total area of the lung section positive for viral RNA.

Computed Tomography (CT) Radiology

CT scans were collected from sedated animals using a 16 slice LightspeedCT scanner (General Electric Healthcare, Milwaukee, Wis., USA) in theprone and supine position. All axial scans were performed at 120 KVp,with Auto mA (ranging between 10 and 120) and were acquired using asmall scan field of view. Rotation speed was 0.8 s. Images weredisplayed as an 11 cm field of view.

To facilitate full examination of the cardiac/pulmonary vasculature,lymph nodes and extrapulmonary tissues post-challenge, Niopam 300(Bracco, Milan, Italy), a non-ionic, iodinated contrast medium, wasadministered intravenously (IV) at 2 ml/kg body weight and scanscollected immediately after injection and ninety seconds from themid-point of injection.

Scans were evaluated for the presence of COVID disease features: groundglass opacity (GGO), consolidation, crazy paving, nodules, peri-lobularconsolidation; distribution—upper, middle, lower, central 2/3,peripheral, bronchocentric) and for pulmonary embolus. The extent oflung involvement was estimated (<25%, 25-50%, 51-75%, 76-100%) andquantified using a scoring system developed for COVID disease.

Results

Analysis of binding titres to either a trimeric form of the S protein orthe isolated receptor binding domain (RBD) showed a small increase inspike (FIG. 17B) and RBD-specific IgG titres (FIG. 17C) in animalsvaccinated with 8 μg after a single vaccination. A greater increase wasobserved in IgG titres after the second vaccination on study day 42,where animals exhibited significant increases with median endpointtitres of 1.6×10³ and 3.2×10³ for S and RBD reactive antibodies,respectively (FIGS. 17B and C). An increase of spike- and RBD specificIgG titres was seen upon challenge in this group, particularly in serumcollected at the time of termination (study days 62, 63 and 64).

As expected, no significant increase in spike or RBD-specific IgGantibodies was seen in the 0.5 μg CVnCoV (intentional sub-optimal dose)or the unvaccinated control group during the vaccination phase. However,a gradual increase in spike- and RBD specific IgG titre was observed ateach of the sampling points in animals vaccinated with 0.5 μg CVnCoVafter challenge (FIGS. 17B and C). No increase in spike- or RBD specificIgG titres was observed in the unvaccinated controls (FIGS. 17B and C).

In agreement with the induction of binding antibodies, robust levels ofvirus neutralising titres (VNTs) were detectable after the secondvaccination in the 8 μg group (FIG. 17D). VNTs peaked on day 42 atmedian titres of 2.7×10⁴. Neutralising antibody titres remainedrelatively unchanged upon challenge until day 62, 63 and 64 of theexperiment. Animals in the 0.5 μg and unvaccinated control groupsremained negative before challenge, while SARS-CoV-2 infection inducedsmall increases in antibody titres in 4/6 and 5/6 animals in the 0.5 μgand unvaccinated group, respectively. Similar results were achieved witha SARS-CoV-2 virus featuring the mutation D614G.

In order to assess CVnCoV induced cellular responses, peripheral bloodmononuclear cells (PBMCs) isolated at different time points postvaccination and challenge were stimulated with pools of peptidesspanning the SARS-CoV-2 spike protein. IFN-γ release of stimulated cellswas measured by ELISpot. Analysis of responses to summed pools in thevaccination phase showed increases in spike-specific IFN-γ in 8 μgCVnCoV vaccinated animals, two weeks after the first and, morepronounced, two weeks after the second vaccination (FIG. 18A panel 1).Stimulation with ten individual pools each covering approx. 140 aminoacids of the S protein demonstrated the induction of cells reactive topeptides across the whole length of S upon vaccination with 8 μg ofCVnCoV (FIG. 18A panel 3).

There were no clear responses in 0.5 μg CVnCoV or unvaccinated animalsin the vaccination phase (FIG. 18A panels 1, 2 and 4). One of the femaleanimals in the negative control group showed particularly high IFN-γsecretion after stimulation with peptide pool 2 (covering part of theN-terminal domain (NTD)) throughout the experiment and peptide pool 3(covering part of the NTD and RBD) on d56 (FIG. 18A panel 1 and 4).

The data demonstrate the strong induction of S specific cellularresponses in CVnCoV vaccinated animals. In animals vaccinated with 8 μgof CVnCoV, increasing responses against peptides covering the wholelength of the S protein were elicited after first and secondvaccination. These data are in line with previous data in mice thatdemonstrated the ability of CVnCoV to induce high S-specific CD4+ andCD8+ T cell responses (e.g. Example 6 and 7). The generation of robust Tcell responses is likely to support vaccine efficacy against SARS-CoV-2.Recent data have demonstrated that CD8+T contribute to viral control ina rhesus macaque model (McMahan et al., 2020). T cell responses toSARS-CoV-2 are readily detectable in humans and may play a role in longterm protection (Grifoni et al., 2020) (Sekine et al., 2020) (Ni et al.,2020).

Increased spike-specific IFN-γ responses were detectable in all animalson day 62-64 post challenge (FIG. 18B panels 1-4). Of note, increases ofcellular responses in animals vaccinated with 8 μg of CVnCoV were lesspronounced than in the other groups, likely indicative of lower levelsof viral replication in these animals (FIG. 18B panel 3).

Quantification of viral RNA copies upon challenge infection demonstrateda reduction of viral replication in the upper respiratory tract in 8 μgCVnCoV vaccinated animals. Importantly, this vaccine dose was able toprotect the lungs of challenged animals. Protection was bothdemonstrated by undetectable levels of viral RNA and by reducedpathological changes upon challenge infection compared to unvaccinatedanimals. Better protection of the lower than of the upper respiratorytract in the presence of robust immune responses is in line with theresults in hamsters (Example 9) and with results of other mRNA basedSARS-CoV-2 vaccines in NHP challenge models (Corbett et al., 2020)(Vogel et al., 2020).

Presence or SARS-CoV-2 total RNA in the upper and lower respiratorytract post-challenge was monitored via qRT-PCR (FIG. 19). Viralreplication in the upper respiratory tract peaked on day 59 inunvaccinated animals, which reached median values of 2.7×107 cp/ml innasal swabs (FIG. 19A) and remained detectable until termination on day62-64. No significant difference between viral replication in animalsvaccinated with 0.5 μg CVnCoV and unvaccinated control animals wasmeasured in nose swabs. Overall, 8 μg CVnCoV vaccination induced thelowest number of viral RNA copies in the upper respiratory tract, wheremedian values of 2.9×10⁶ cp/ml in nasal swabs, respectively, weredetectable on day 59. However, the difference between the study groupswas not statistically significant. Comparable results were generated inthroat swabs (FIG. 20A).

Additional analyses assessing subgenomic (sg) RNA via qRT PCR indicativeof viral replication yielded overall low sgRNA levels in the upperrespiratory tract. Values peaked on day 59 and returned to baseline onday 62 in all animals. In nasal swabs, sg RNA levels were lowest inCVnCoV vaccinated animals with values of 0.4×10⁴ compared to 3.7×10⁴cp/ml in unvaccinated control animals. 3 of 6 animals in the 8 μg CVnCoVvaccinated group remained negative at all time points while 5/6 animalsin the unvaccinated group had detectable levels of subgenomic viral RNA(FIG. 19D). Analyses of throat swabs showed no significant difference ofsubgenomic RNA between the groups and median values remained below thelower limit of quantification in all animals (FIG. 20B).

Parallel analyses of the lower respiratory tract of in life (d59) andpost-mortem (d62-d64) bronchoalveolar lavage (BAL) samples showedsignificantly reduced levels of total viral RNA upon 8 μg CVnCoVvaccination at both time points (FIG. 19B). Median values of total RNAon day 59 and day 62-64 were 4.3×10⁶ and 1.1×10⁵ cp/ml in the controlgroup, while animals vaccinated with 8 μg of CVnCoV featured mediantitres of 0.6×10⁴ and 0.3×10⁴, respectively. RNA levels in BAL werebelow the lower limit of quantification for all but one animal in the 8μg CVnCoV group on day 59, which featured low RNA counts. Total viralRNA levels in 0.5 μg CVnCoV vaccinated animals were comparable to thecontrol group. Of note, BAL analyses on day 59 only depict femaleanimals and one male animal of the unvaccinated group. The remaininganimals were excluded from this analysis since suboptimal BAL samplingconditions had been chosen that prevented further evaluation.

The analysis of lung tissue collected at necropsy confirmed resultsgained in BAL samples. Median titres of 2.9×10⁸ cp/g were detectable inthe unvaccinated group while all animals in the CVnCoV 8 μg vaccinatedgroups remained below the lower limit of quantification (FIG. 19C).There was no statistically significant difference between animals in the0.5 μg CVnCoV and the unvaccinated group.

In terms of vaccine safety, the injection of 0.5 μg or 8 μg of CVnCoVelicited no adverse reactions to vaccination and no differences inweight or temperature were observed between groups during thevaccination phase of the study (data not shown), supporting a favourablesafety profile of the vaccine in rhesus macaques at the doses used.Furthermore, no signs of vaccine enhanced disease were detectable inthis study.

Subgenomic viral RNA analysis in BAL and lung tissue samples yieldedcomparable results: RNA indicative of replicating virus were detectablein BAL and lung samples of unvaccinated and 0.5 μg CVnCoV vaccinatedanimals on day 59 and day 62-64, respectively. All animals in the 8 μgCVnCoV group were negative in these analyses (FIGS. 19E and F).

Evaluation of further tissue samples collected at necropsy revealed lowbut detectable signals of SARS-CoV-2 total RNA in trachea and tonsils of0.5 μg CVnCoV and unvaccinated animals, while 8 μg CVnCoV vaccinatedanimals remained negative (FIGS. 20C and D). No viral RNA was detectablein spleen, duodenum, colon, liver or kidney in any group (FIG. 20E-I).

Histopathological analyses of lung samples taken at necropsy showedlesions consistent with infection with SARS-CoV-2 in the lungs ofchallenged animals (FIG. 21). Briefly, the lung parenchyma showedmultifocal to coalescing areas of pneumonia surrounded by unaffectedparenchyma. Alveolar damage, with necrosis of pneumocytes was aprominent feature in the affected areas. The alveolar spaces withinthese areas were often thickened and damaged alveolar walls containedmixed inflammatory cells (including macrophages, lymphocytes, viable anddegenerated neutrophils, and occasional eosinophils). Alveolar oedemaand alveolar type II pneumocyte hyperplasia was also observed. In distalbronchioles and bronchiolo-alveolar junctions, degeneration andsloughing of epithelial cells was present. In the larger airwaysoccasional, focal, epithelial degeneration and sloughing was observed inthe respiratory epithelium. Low numbers of mixed inflammatory cells,comprising neutrophils, lymphoid cells, and occasional eosinophils,infiltrated bronchial and bronchiolar walls. In the lumen of someairways, mucus admixed with degenerated cells, mainly neutrophils andepithelial cells, was seen. Within the parenchyma, perivascular andperibronchiolar cuffing was also observed, being mostly lymphoid cellscomprising the infiltrates. No remarkable changes were observed innon-pulmonary tissues.

In agreement with reduced levels of viral RNA, the evaluation of lungsamples using a histopathology scoring system showed a significantreduction in severity of lung lesions in CVnCoV vaccinated animalscompared to 0.5 μg CVnCoV vaccinated and unvaccinated groups (FIGS. 22Aand B).

Viral RNA was observed in alveolar epithelia cells and within theinflammatory cell infiltrates (FIG. 21). The quantity of virus RNAobserved by in situ hybridisation (ISH) was also significantly reducedin 8 μg CVnCoV vaccinated animals when compared to 0.5 μg CVnCoV andunvaccinated (FIG. 22C).

In order to gain an in-life view of pathological changes induced uponSARS-CoV-2 infection in the complete lung, CT scanning was performedprior to challenge and post-challenge on study day 61. Overall, theapparent level of disease was relatively mild and only affected lessthan 25% of the lung. Post-challenge, abnormalities in the lung weredetected in 6 of 6 animals the 0.5 μg CVnCoV group, and 5 of 6 in theunvaccinated control group, while only 3/6 animals vaccinated with 8 μgCVnCoV exhibited detectable changes. Lowest levels of total scoring inCT scans were seen in animals vaccinated with 8 μg of CVnCoV (FIG. 22D).Of note, highest scores were seen in the 0.5 μg CVnCoV group in thisanalysis. However, values were not statistically different to thecontrol group.

No indication of enhanced disease was detectable upon assessment ofclinical signs post-challenge and the compositions were found to behighly immunogenic in rhesus macaques. There were no clear differencesin body weight or temperature between groups post-challenge or any signsof fever. Vaccine enhanced disease can be caused by antibodies(antibody-dependent enhanced disease, ADE (reviewed in (Lee et al.,2020)) as previously described for a feline coronavirus (Olsen et al.,1992). Such antibodies most likely possess non-neutralising activity,and enhance viral entry causing increased viral replication and diseaseexacerbation. Results presented here give no indication of increasedviral replication in animals vaccinated with CVnCoV. Importantly,enhanced replication in the respiratory tract or distal organs such asspleen, duodenum, colon, liver or kidney was also not detectable in the0.5 μg group of the study. These animals featured low levels of Sbinding but undetectable levels of VNTs upon challenge infection,creating conditions under which ADE could hypothetically can occur.

Another cause of disease enhancement may be vaccine-associated enhancedrespiratory disease (VAERD) that is hallmarked by increased inflammationdue to TH2-biased immune responses and high ratios of non-neutralisingto neutralising antibodies (reviewed in (Graham, 2020), (Lee et al.,2020), (Smatti et al., 2018). Analysis of lung pathology in CVnCoVvaccinated animals demonstrated protectivity of 8 μg CVnCoV and gave noindication for increased inflammation and pathological changes insuboptimally dosed animals.

The results extend our knowledge of CVnCoV safety, immunogenicity andprotective efficacy in a highly relevant model system for SARS-CoV-2.The overall outcome of the study in non-human primates in terms ofimmunogenicity, protective efficacy and pathology are comparable toresults in the hamster model (see Example 9), providing support forhamsters as a models system for SARS-CoV. Therefore, CVnCoV is highlyefficacious at a low dose of 8 μg in a COVID-19 NHP challenge modelwhile being safe at both doses tested with lack of any indication ofdisease enhancement.

In another similar NHP study vaccine composition comprising mRNAencoding S_stab formulated in LNPs comprising the inventive form of the3′ end (hSL-A100) and UTR combination (a-1 (HSD17B4/PSMB3)) (R9709) isanalysed and compared to CVnCoV (R9515).

Example 16: Vaccination of Mice with mRNA Encoding SARS-CoV-2 AntigenS_Stab Formulated in LNPs

The present example shows that SARS-CoV-2 S mRNA vaccines with mRNAcomprising improved non coding regions induce strong immune responses.Some further groups of mice received mRNA vaccine composition comprisingchemically modified nucleotides (N(1)-methylpseudouridine, m1ψ). Onegroup of mice received an mRNA vaccine composition comprising mRNAproduced with an alternative Cap (3′OME Clean Cap). More details areindicated in Table 22.

Preparation of LNP Formulated mRNA Vaccine:

SARS-CoV-2 S mRNA constructs are prepared as described in Example 1 (RNAin vitro transcription). HPLC purified mRNA was formulated with LNPsaccording to Example 1.4.

Immunostimulation of Human Peripheral Blood Mononuclear Cells (PBMCs)

Preparation of Human PBMCs

Human peripheral blood mononuclear cells (PBMCs) were isolated fromwhole blood of anonymous donors by standard Ficoll-Hypaque densitygradient centrifugation (Ficoll 1.078 g/ml). PBMCs were washed with PBSand re-suspended in RPMI 1640 supplemented with 20% heat-inactivatedFCS, 1% Penicillin/Streptomycin and 1% L-Glutamine. After counting,cells are re-suspended at 50 million cells per ml in fetal calf serum,10% DMSO, and frozen. Before usage, the cells are thawed.

PBMC Stimulation

PBMC were stimulated with 10 μg/ml of LNP-formulated mRNA (Table 22, rowB-M) at a density of 4×10⁵ cells in a total volume of 200 μl in ahumidified 5% CO₂ atmosphere at 37° C. To quantify backgroundstimulation, PBMC were incubated with medium only (Table 22, row A). 24hours after transfection, supernatants were collected.

Quantification of Cytokine Levels

Human IFNα was quantified using an IFNα ELISA from PBL according tomanufacturer's instructions. PBMC supernatants were used in a 1:20 or1:40 dilution and 50 μl of the dilution are added to 50 μl prefilledbuffer.

Immunization:

Female BALB/c mice (6-8 weeks old, n=8) were injected intramuscularly(i.m.) with mRNA vaccine compositions indicated in Table 22. As anegative control, one group of mice was vaccinated with buffer (groupA). All animals were vaccinated on day 0 and 21. Blood samples werecollected on day 21 (post prime) and 42 (post boost) for thedetermination of antibody titers, splenocytes were isolated on day 42for T-cell analysis.

TABLE 22 Vaccination regimen (Example 16): 5′-UTR/ 3′-UTR; SEQ SEQ Mod.Vaccine mRNA CDS UTR ID NO: ID NO: nucleo- Group composition ID opt.Design 3′-end Protein RNA Dose tides A buffer — — — — — B mRNA encodingR9515  opt1 —/muag A64-N5- 10 163 1 μg — S_stab formulated C30-hSL-N5 inLNPs C mRNA encoding R9709  opt1 HSD17B4/ hSL-A100 10 149 1 μg — S_stabformulated PSMB3 in LNPs D mRNA encoding R10153  opt1 HSD17B4/ A100 1024837 1 μg — S_stab formulated PSMB3 in LNPs E mRNA encoding R10154 opt1 —/muag A100 10 25717 1 μg — S_stab formulated in LNPs F mRNAencoding R10155  opt1 Rpl31/RPS9 hSL-A100 10 23957 1 μg — S_stabformulated in LNPs G mRNA encoding R10156  opt1 Rpl31/RPS9 A100 10 252771 μg — S_stab formulated in LNPs H mRNA encoding R10157  opt1 —/muagA64-N5- 10 163 1 μg m1ψ S_stab formulated C30-hSL-N5 in LNPs I mRNAencoding R10158  opt1 —/muag hSL-A100 10 24397 1 μg m1ψ S_stabformulated in LNPs J mRNA encoding R10159  opt1 HSD17B4/ hSL-A100 10 1491 μg m1ψ S_stab formulated PSMB3 in LNPs K mRNA encoding R10160* opt1HSD17B4/ hSL-A100 10 149 1 μg — S_stab formulated PSMB3 in LNPs L mRNAencoding R10161* opt1 HSD17B4/ hSL-A100 1 148 1 μg — S formulated inPSMB3 LNPs M mRNA encoding R10162  opt10 HSD17B4/ hSL-A100 10 151 1 μgm1ψ S_stab formulated PSMB3 in LNPs *mRNA R10160 (group K) and R10161(groupL) were produced with 3′OME Clean Cap.

Determination of IgG1 and IgG2 antibody titers using ELISA and virusneutralizing titers via CPE (cytopathic effect) were performed asdescribed in Example 6. T-cell analysis by Intracellular cytokinestaining (ICS) are performed as described in Example 6.

Results:

As shown in FIG. 23A mRNA encoding full length S stabilized protein(S_stab) induced different levels of IFNα in human PBMCs. For most ofthe constructs moderate levels of IFNα were induced, whereasLNP-formulated mRNA comprising chemically modified nucleotides did notinduce IFNa.

The vaccination with mRNA encoding full length S stabilized protein(S_stab) comprising improved non-coding regions induced strong levels ofvirus neutralizing antibody titers (VNTs) already on day 21 after firstvaccination (shown in FIG. 23B). All of the mRNA vaccine compositionswith mRNAs comprising a 3′ end “hSL-A100” or “A-100” (groups C-G, I-M)showed this improved, early and strong induction of VNTs. In theseconstructs, the poly(A) sequence is located directly at the 3′ terminusof the RNA.

The introduction of chemically modified nucleotides (groups H, I, J, M)led to comparable VNTs.

After the second vaccination on day 42 (shown in FIG. 23C), most of themRNA vaccines show a robust induction of high titers of VNTs. AlsoCVnCoV, vaccine composition comprising mRNA with the 3′ endA64-N5-C30-hSL-N5 (group B) induced very high amount of VNT. Theintroduction of chemically modified nucleotides (m1ψ, group H) resultedin decreased levels. All of the mRNA vaccine compositions with mRNAscomprising a 3′ end “hSL-A100” or “A-100” (groups C-G, I-M) showed thisimproved, early and strong induction of VNTs, irrespectively of usingm1ψ or not. In these constructs, the poly(A) sequence is locateddirectly at the 3′ terminus of the RNA.

Example 17: Vaccination of Mice with mRNA Encoding SARS-CoV-2 AntigenS_Stab Formulated in LNPs

The present example shows that SARS-CoV-2 S mRNA vaccines with mRNAcomprising improved non coding regions induce strong immune responses.This study compares an “only prime” with a “prime-boost” vaccinationregimen. Some groups of mice received mRNA vaccine compositioncomprising chemically modified nucleotides (N(1)-methylpseudouridine,m1ψ). More details are indicated in Table 23.

Preparation of LNP Formulated mRNA Vaccine:

SARS-CoV-2 S mRNA constructs are prepared as described in Example 1 (RNAin vitro transcription). HPLC purified mRNA was formulated with LNPsaccording to Example 1.4.

Immunization:

Female BALB/c mice (6-8 weeks old, n=8) were injected intramuscularly(i.m.) with mRNA vaccine compositions indicated in Table 23. As anegative control, one group of mice was vaccinated with buffer (groupK). The animals were vaccinated only one time on day 0 (group A-E), ortwice on day 0 and 21 (group F-J). Blood samples were collected on day21 and 42 for the determination of antibody titers, splenocytes wereisolated on day 42 for T-cell analysis.

TABLE 23 Vaccination regimen (Example 17): 5′-UTR/ 3′-UTR; SEQ SEQ Mod.Vaccine mRNA CDS UTR ID NO: ID NO: nucleo- Group composition ID opt.Design 3′-end Protein RNA Dose tides A mRNA encoding R9515  opt1 —/muagA64-N5- 10 163 1 μg — S_stab formulated C30-hSL- day 0 in LNPs N5 B mRNAencoding R9709  opt1 HSD17B4/ hSL- 10 149 1 μg — S_stab formulated PSMB3A100 day 0 in LNPs C mRNA encoding R10157 opt1 —/muag A64-N5- 10 163 1μg m1ψ S_stab formulated C30-hSL- day 0 in LNPs N5 D mRNA encodingR10159 opt1 HSD17B4/ hSL- 10 149 1 μg m1ψ S_stab formulated PSMB3 A100day 0 in LNPs E mRNA encoding R10162 opt10 HSD17B4/ hSL- 10 151 1 μg m1ψS_stab formulated PSMB3 A100 day 0 in LNPs F mRNA encoding R9515  opt1—/muag A64-N5- 10 163 1 μg — S_stab formulated C30-hSL- day 0 in LNPs N5day 21 G mRNA encoding R9709  opt1 HSD17B4/ hSL- 10 149 1 μg — S_stabformulated PSMB3 A100 day 0 in LNPs day 21 H mRNA encoding R10157 opt1—/muag A64-N5- 10 163 1 μg m1ψ S_stab formulated C30-hSL- day 0 in LNPsN5 day 21 I mRNA encoding R10159 opt1 HSD17B4/ hSL- 10 149 1 μg m1ψS_stab formulated PSMB3 A100 day 0 in LNPs day 21 J mRNA encoding R10162opt10 HSD17B4/ hSL- 10 151 1 μg m1ψ S_stab formulated PSMB3 A100 day 0in LNPs day 21 K buffer — — — — day 0 — day 21

Determination of virus neutralizing titers via CPE (cytopathic effect)were performed as described in Example 6. T-cell analysis byIntracellular cytokine staining (ICS) are performed as described inExample 6.

Results:

FIG. 24A shows the induction of VNTs after only one vaccination. Asshown before in Example 17. mRNA vaccine compositions with mRNAscomprising a 3′ end “hSL-A100” or “A-100” (groups A, D, E, G, I, and J))showed improved, early and strong induction of VNTs. In theseconstructs, the poly(A) sequence is located directly at the 3′ terminusof the RNA.

FIG. 24B demonstrate the induction of VNTs after only one vaccination(group A-E) or after two vaccination (group F-J) on day 42. mRNA vaccinecomposition comprising R9709 (group B) induced most prominent titers ofVNTs between the groups receiving only one vaccination. The strength ofvaccine composition comprising R9709 may support an immunizationprotocol for the treatment or prophylaxis of a subject againstcoronavirus, preferably SARS-CoV-2 coronavirus comprising only onesingle dose of the composition or the vaccine. mRNA vaccine compositionswith mRNAs comprising a 3′ end “hSL-A100” or “A-100” (groups A, D, E, G,I, and J)) showed improved, early and strong induction of VNTs. In theseconstructs, the poly(A) sequence is located directly at the 3′ terminusof the RNA.

Example 18: Efficacy of mRNA Vaccines in K18-hACE2 Mouse Model forSARS-CoV-2 Infection

Mice are not susceptible to infection with SARS-CoV-2, but a geneticallyengineered mouse model has been developed that expresses the humanreceptor ACE2 (hACE2), required for entry of the virus into the hostcell under the K18 promoter. The model was originally developed toinvestigate the causative agent of SARS (SARS-CoV) (MCCRAY, Paul B., etal. Lethal infection of K18-hACE2 mice infected with severe acuterespiratory syndrome coronavirus. Journal of virology, 2007, 81. Jg.,Nr. 2, S. 813-821) but is now also used as a suitable small animal modelfor COVID-19. Previously, hACE2 mice have been shown to be susceptibleto SARS-CoV-2 and to exhibit a disease course with weight loss,pulmonary pathology, and symptoms similar to those in humans (e.g. BAO,Linlin, et al. The pathogenicity of SARS-CoV-2 in hACE2 transgenic mice.Nature, 2020, 583. Jg., Nr. 7818, S. 830-833, or YINDA, Claude Kwe, etal. K18-hACE2 mice develop respiratory disease resembling severeCOVID-19. PLoS pathogens, 2021, 17. Jg., Nr. 1, S. e1009195; DE ALWIS,Ruklanthi M., et al. A Single Dose of Self-Transcribing and ReplicatingRNA Based SARS-CoV-2 Vaccine Produces Protective Adaptive Immunity InMice. BioRxiv, 2020). In principle, the K18-hACE2 mouse is suitable forvaccine studies to investigate the prevention of infection withSARS-CoV-2 or the reduction of viral load, and at the same time toinvestigate the correlates and causes of a protective effect of an mRNAvaccine against COVID-19 with well-established immunological methods,which are generally available for mouse models.

The present example shows that SARS-CoV-2 S mRNA vaccines induce stronghumoral as well as cellular immune response in K18-hACE2 mice.SARS-CoV-2 S mRNA vaccines protect K18-hACE2 mice from SARS-CoV-2 viralchallenge, which can be shown e.g. by measuring the viral loads ofinfected animals, by monitoring the disease progression with weightloss, pulmonary pathology and other symptoms, or by histopathology andsurvival.

Preparation of LNP Formulated mRNA Vaccine:

SARS-CoV-2 S mRNA constructs are prepared as described in Example 1 (RNAin vitro transcription). HPLC purified mRNA was formulated with LNPsaccording to Example 1.4 prior to use in in vivo vaccinationexperiments.

Immunization and Challenge:

K18-hACE2 mice are injected intramuscularly (i.m.) with mRNA vaccinecompositions and doses as indicated in Table 24. As a negative control,one group is vaccinated with buffer. As a control, one group is injectedintramuscularly with, 20 μl Formalin-inactivated SARS-CoV-2 virus (10⁶PFU) adjuvanted with Alum. All animals were vaccinated on day 0 and day28. Blood samples were collected on day 0, day 28 (post prime) and 56(post boost, before challenge) for the determination of antibody titers.The animals are challenged intranasally with e.g. 10⁵ PFU SARS-CoV-2(Bavaria 1) at day 56. Animals were followed for four to ten days postchallenge.

TABLE 24 Vaccination regimen (Example 18): 5′-UTR/ 3′-UTR; SEQ SEQ mRNACDS UTR ID NO: ID NO: Group Vaccine composition ID opt. Design 3′-endProtein RNA Dose A buffer — — — — 20 μL B mRNA encoding S_stab R9515opt1 —/muag; A64-N5- 10 163   8 μg formulated in LNPs C30-hSL-N5 C mRNAencoding S_stab R9709 opt1 HSD17B4/ hSL-A100 10 149   8 μg formulated inLNPs PSMB3 D mRNA encoding S_stab R9709 opt1 HSD17B4/ hSL-A100 10 149  2 μg formulated in LNPs PSMB3 E mRNA encoding S_stab R9709 opt1HSD17B4/ hSL-A100 10 149 0.5 μg formulated in LNPs PSMB3 FFormalin-inactivated — — — — — — 10⁶ PFU, virus + Alum 20 μL,

Determination of IgG1 and IgG2 antibody titers using ELISA,determination of virus neutralizing titers via CPE (cytopathiceffect)-based microneutralization assay and T-cell analysis byIntracellular cytokine staining (ICS) are performed as described inExample 6.

The immunization and challenge of K18-hACE2 mice as described in thepresent Example can be used to determine the protective efficacy offurther inventive mRNA constructs and compositions. Furthermore, byusing mutated virus variants or isolates of SARS-CoV-2 (e.g. B.1.351,see also Table 25), it can be shown, that the inventive mRNA vaccinecompositions are effective in addition against these mutated virusvariants or isolates.

Example 19: Neutralizing Activity of mRNA Vaccines Against EmergingSARS-CoV-2 Variants

The neutralizing activity of sera from 20 phase 1 clinical trialparticipants (aged 18-60 years, male and female) who received two dosesof 2 μg, 4 μg, 8 μg, or 12 μg CVnCoV (see Example 10 regarding theclinical study outline) is tested against emerging SARS-CoV-2 variantsor isolates. The serum samples were obtained on days 36, 43 or 57 of theclinical study and exhibit Virus neutralizing antibody titers (VNTs) ata range of 10-1280 (MN 25TCID50), representing samples with low (10-20)and with high (452-1280) VNTs.

VNTs are analysed as described e.g. for the analysis of hamster sera inExample 9, whereby the serum samples are incubated with emerging virusvariants. Strain SARS-CoV-2/human/ITA/INMI1/2020 orUVE/SARS-CoV-2/2020/FR/702 can be used as reference (“wildtype strain”).Emerging SARS-CoV-2 variants or isolates for analysis are listed inTable 25. Further variants may arise and can be tested as well.

TABLE 25 List of emerging SARS-CoV-2 variants (Example 19): Amino acidchanges Variant in spike protein Mink Cluster 5 variant GISAID: delH69,delV70, Y453F, EPI_ISL_616802 (hCoV- D614G, I692V, M1229I19/Denmark/DCGC-3024/2020) B.1.1.7 (a.k.a., 20B/501Y.V1, delH69, delV70,delY144, 501Y.V1, Variant of Concern- N501Y, A570D, D614G, 202012/01,VOC-202012/01, P681H, T716I, S982A, D1118H VUI-202012/01, B117, “UKvariant”) B.1.351 (a.k.a., 20C/501Y.V2, L18F, D80A, D215G, 501Y.V2,N501Y.V2, “SA delL242, delA243, delL244, variant”, “South Africavariant”) R246I, K417N, E484K, N501Y, D614G, A701V P.1 (a.k.a., “Brazilvariant” = L18F, T20N, P26S, D138Y, “Japan variant”) R190S, K417T,E484K, N501Y, D614G, H655Y, T1027I CAL.20C (a.k.a., “Californiavariant”) S13I, W152C, L452R, D614G NCBI: QQN00429.1 (CAL.20.C example:SARS-CoV- 2/human/USA/CA- LACPHL-AF00114/2021)

Neutralization can also be measured by a recombinant VSV- orlentiviral-based pseudovirus neutralization (PsVN) assay thatincorporates the spike mutations present in the SARS-CoV-2 variantstrain.

The capacity of binding antibodies can be tested in ELISA assays asdescribed above in e.g. Example 10 with recombinant spike proteins forcoating featuring the mutations of emerging virus variant.

The efficacy of mRNA vaccine can also be tested in challenge models asdescribed e.g. in Example 17 for hACE-mice, in Example 15 for NHPs, andin Example 9 for hamsters using an emerging SARS-Cov-2 variant for thechallenge infection.

1. A method of stimulating an immune response in a subject, the methodcomprising administering to the subject an effective amount of acomposition comprising: (I) a mRNA comprising: (a) at least one codingsequence encoding a SARS-CoV-2 spike protein (S) at least 90% identicalto SEQ ID NO: 10 that is a pre-fusion stabilized spike protein (S_stab)comprising a pre-fusion stabilizing K986P and V987P mutation; and (b) atleast one heterologous untranslated region (UTR); and (II) at least onepharmaceutically acceptable carrier, wherein the mRNA is complexed withlipid nanoparticles (LNP) and wherein the LNP comprise: (i) at least onecationic lipid according to formula III-3:

(ii) at least one neutral lipid, comprising1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC); (iii) at least onesteroid, comprising cholesterol; and (iv) at least one PEG-lipidaccording to formula IVa:

wherein (i) to (iv) are in a molar ratio of about 20-60% cationic lipid,5-25% neutral lipid, 25-55% sterol, and 0.5-5% PEG-lipid, wherein thecomposition is administered by intramuscular injection.
 2. The method ofclaim 1, wherein the method produces coronavirus neutralizing antibodiesin the subject.
 3. The method of claim 1, wherein the mRNA comprises atleast one coding sequence encoding a SARS-CoV-2 spike protein (S) atleast 95% identical to SEQ ID NO:
 10. 4. The method of claim 3, whereinthe mRNA comprises a 5′-cap structure and at least one poly(A) sequencecomprising 30 to 200 adenosine nucleotides.
 5. The method of claim 4,wherein 5′-cap structure comprises a m7G, cap0, cap1, cap2, a modifiedcap0, or a modified cap1 structure.
 6. The method of claim 4, whereinthe at least one coding sequence of the mRNA is a codon modified codingsequence, wherein the at least one codon modified coding sequence isselected from the group consisting of C maximized coding sequence, CAImaximized coding sequence, human codon usage adapted coding sequence,G/C content modified coding sequence, and G/C optimized coding sequence,or any combination thereof.
 7. The method of claim 4, wherein the atleast one coding sequence of the mRNA has a G/C content that isincreased by at least 10% compared to the G/C content of the codingsequence of the corresponding reference nucleic acid sequence.
 8. Themethod of claim 4, wherein the mRNA comprises a sequence at least 80%identical to SEQ ID NO:
 137. 9. The method of claim 8, wherein the mRNAcomprises a sequence at least 90% identical to SEQ ID NO:
 137. 10. Themethod of claim 4, wherein the at least one heterologous untranslatedregion is selected from at least one heterologous 5′-UTR and/or at leastone heterologous 3′-UTR.
 11. The method of claim 10, wherein the atleast one heterologous 3′-UTR comprises or consists of a nucleic acidsequence derived from a 3′-UTR of a gene selected from the groupconsisting of PSMB3, ALB7, alpha-globin, CASP1, COX6B1, GNAS, NDUFA1,and RPS9.
 12. The method of claim 10, wherein the at least oneheterologous 5′-UTR comprises or consists of a nucleic acid sequencederived from a 5′-UTR of a gene selected from the group consisting ofHSD17B4, RPL32, ASAH1, ATP5A1, MP68, NDUFA4, NOSIP, RPL31, SLC7A3,TUBB4B, and UBQLN2.
 13. The method of claim 4, wherein the nucleic acidcomprises at least one histone stem-loop.
 14. The method of claim 4,wherein 100% of the uracil positions in the at least one coding sequenceof the mRNA have a chemical modification.
 15. The method of claim 4,wherein 100% of the uracil positions in the at least one coding sequenceof the mRNA are substituted with pseudouridine orN(1)-methylpseudouridine.
 16. The method of claim 15, wherein 100% ofthe uracil positions in the at least one coding sequence of the mRNA aresubstituted with N(1)-methylpseudouridine.
 17. The method of claim 4,wherein the mRNA has been purified by a method comprising TFF.
 18. Themethod of claim 17, wherein the mRNA has been purified by a methodcomprising TFF and RP-HPLC.
 19. (canceled)
 20. The method of claim 16,wherein the LNP comprise a PEG lipid according to formula IVa:

and wherein n has a mean value ranging from about 30 to
 60. 21. Themethod of claim 20, wherein the LNP comprise at least one PEG-lipidaccording to formula (IVa) wherein n has a mean value ranging from about30±2, 32±2, 34±2, 36±2, 38±2, 40±2, 42±2, 44±2, 46±2, 48±2, 50±2, 52±2,54±2, 56±2, 58±2, or 60±2.
 22. The method of claim 20, wherein thecomposition further comprises a lyoprotectant.
 23. The method of claim22, wherein the lyoprotectant comprises sucrose.
 24. The method of claim20, wherein the composition has a lipid to RNA molar ratio (N/P ratio)of from about 2 to about
 12. 25. The method of claim 20, wherein the LNPhave a mean diameter of from about 60 nm to about 200 nm.
 26. The methodof claim 25, wherein the LNP comprise components (i) to (iv) in a molarratio of approximately 50:10:38.5:1.5 (cationic lipid:neutrallipid:cholesterol:PEG-lipid). 27-28. (canceled)
 29. The method of claim25, wherein the composition comprises about 1 μg to about 200 μg of themRNA.
 30. The method of claim 29, wherein the composition comprisesabout 5 μg to about 100 μg of the mRNA.
 31. The method of claim 29,wherein the method comprises administering the composition to thesubject at least twice.
 32. A method of stimulating an immune responsein a subject comprising administering to the subject an effective amountof a composition comprising: (I) a mRNA comprising from a 5′ to 3′: (a)the mRNA comprises a 5′-cap structure; (b) at least one coding sequenceencoding a SARS-CoV-2 spike protein (S) at least 95% identical to SEQ IDNO: 10 that is a pre-fusion stabilized spike protein (S_stab) comprisinga pre-fusion stabilizing K986P and V987P mutation, wherein 100% of theuracil positions in the at least one coding sequence of the mRNA aresubstituted with N(1)-methylpseudouridine; (c) a heterologous 3′ UTR;and (d) at least one poly(A) sequence comprising 30 to 200 adenosinenucleotides; and (II) at least one pharmaceutically acceptable carrier,wherein the mRNA is complexed with lipid nanoparticles (LNP) and whereinthe LNP comprises: (i) at least one cationic lipid according to formulaIII-3:

(ii) 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC); (iii)cholesterol; and (iv) at least one PEG-lipid according to formula IVa:

wherein (i) to (iv) are in a molar ratio of about 20-60% cationic lipid,5-25% DSPC, 25-55% cholesterol, and 0.5% to 5% PEG-lipid, wherein thecomposition is administered by intramuscular injection.
 33. The methodof claim 32, further defined as a method of stimulating production ofSARS-CoV-2 spike protein-binding antibodies in the subject.